JP2008532474A - Tissue modeling in embryonic stem (ES) cell lines - Google Patents

Abstract

An embryonic stem (ES) cell-derived tissue modeling system is provided. Specifically, a system for de novo production of tissue by selecting parallel drugs in one cultured strain of ES cells differentiating cell types constituting the target tissue, Explain the use in transplantation and drug development.

Description

Field of Invention

  The present invention generally relates to the use of embryonic and embryonic stem cell derived cell types suitable for use in tissue regeneration and non-therapeutic applications such as drug screening.

Background Art Progenitor cells have become a central concern in medical research. Numerous tissues in the body have a backup reserve of precursors that can replace cells that are aged or damaged by injury or disease. Considerable efforts have recently been made to isolate a number of different tissue precursors for use in regenerative medicine. Sources and systems for generating differentiated cells for use from stem cell populations when a relatively homogeneous cell population is desired are summarized, for example, in US Patent Application US2003 / 0040111. Mammalian pluripotent and multipotent embryonic stem (ES) cells and embryonic germ (EG) cells can be induced in culture to differentiate into a variety of cell types including cardiomyocytes. However, ES cell-derived cardiomyocytes constitute only 1% to 5% of all cells of the embryoid body (EB) after differentiation. Most of them consist of undifferentiated ES cells with a high likelihood of tumor formation.

  Recently, methods have been described for genetic selection of specific cell types from embryonic stem (ES) cell differentiating cultures based on the use of tissue-specific gene regulatory sequences, promoters that drive drug resistance cassettes; See International Application WO02 / 051987. In this way, specific differentiated cell types generated from transgenic ES clones with corresponding vectors are selected by using corresponding drugs that eradicate other generated cell types and undifferentiated ES cells. We were able to. To date, this method has proven to be the most specific and efficient for highly purifying heart, nerve and insulin secreting cells from differentiating ES cell cultures.

  However, a major challenge in using stem cells for treatment is to control the growth and differentiation into specific tissue types necessary for treatment of each patient.

  Thus, there is a need for new approaches to create a population of differentiated cells and tissues suitable for human administration. The solution to the above technical problem is achieved by providing the embodiments characterized in the claims and further described below.

SUMMARY OF THE INVENTION All tissues, together with supporting cell types (eg, fibroblasts, stromal cells, endothelial cells, glial cells, etc.), the three-dimensional constitutive structure of a tissue, its nutritional functions, and organisms It is known to be composed of the main specific cell types that determine their functional role that is important for maintaining interconnection with the entire other tissue system.

  The present invention shows that the layout of most of the tissues that make up an adult organism is such that during the development of the early embryo, the corresponding cell types appear during differentiation and interact with each other according to specific signaling molecules and the developing receptors. It is based on the theory that it is established when a connection is formed. Thus, when various cell types contributing to a particular tissue type are genetically selected from the same differentiating ES cell culture lines, these are their natural, genetically determined It can be anticipated that interconnections and configurations must be formed according to specific pathways. In such cases, it is possible to purify the target cells at a high level among the differentiating strains of the genetically modified ES cells, and to “self-assemble” the tissue-like structure in the course of ES cell differentiation in vitro. It is the main precondition for making it happen.

  According to the present invention, when ES cell-derived cardiomyocytes are co-cultured and co-transplanted with embryonic fibroblasts, cardiac tissue-like formation occurs in vitro, and the transplantation results are significantly improved when injected into a cold infarct heart of mice I was able to show surprisingly that

  Thus, in one aspect, the present invention comprises culturing a first cell type derived from embryonic stem (ES) cells in the presence of at least one embryonic second cell type; and the at least two cell types; Integrating and aligning them into a tissue or tissue-like structure, wherein in this case, preferably, the ES cell of the first cell type derived from the ES cell is a first cell type specific for the first cell type. It relates to a method for modeling and / or obtaining a tissue or tissue-like structure comprising a selection marker operatively linked to a cell type specific regulatory sequence. Therefore, the use of a highly efficient drug selection system effectively increases the final yield (5 to 10 times) due to the intensive cardiomyocyte proliferation, and there is a risk of tumor development after transplantation. Decreases to an unsatisfactory level.

In accordance with the above, the present invention generally
(A) Introducing a cell inoculum containing a co-cultured strain of an ES cell-derived cell type and embryonic feeder cells that has undergone differentiation into at least a part of a previously damaged tissue area, or a differentiated tissue With the steps to introduce:
(B) transplanting the introduced cellular inoculum in situ as viable cells or tissues placed in the previously damaged tissue section, wherein the feeding results in the feeding Improving the tissue repair and / or organ function in a mammal, comprising improving the tissue and / or organ function of the animal.

  Said feeder cells are preferably fibroblasts and / or endothelial cells.

Specifically, a method is provided for improving cardiac function in a mammal after myocardial infarction, said method comprising:
(A) An undifferentiated mammalian embryonic stem (ES) cell containing a resistance gene and a reporter gene under the control of the same heart-specific promoter is cultured in vitro in a medium containing a selective agent for the resistance gene. And culturing under conditions that allow differentiation of the ES cells into cardiomyocytes;
(B) isolating said differentiated cardiomyocytes and / or pre-differentiated cells, optionally together with differentiating cells from said cardiomyocytes towards an irrelevant cell type in the course of differentiation Removing the step;
(C) then co-implanting the cardiomyocytes with embryonic or ES cell-derived fibroblasts and / or endothelial cells into at least a portion of a previously infarcted area of heart tissue;
(D) transplanting the introduced cellular inoculum in situ as viable cells placed in a previously infarcted area in the heart tissue, wherein the transplantation results in the mammalian Improving the cardiac function.

  It is to be understood that for actual transplantation, the co-transplantation of the cells need not be performed simultaneously, and may be performed in any order.

  Embryonic cells are not always available as a source of support for ES cell-derived cell types to develop into specific tissues, or specific embryonic cells may not be suitable for this purpose. Furthermore, there may be other reasons why the use of these cells is not suitable, for example, the state of cell development is different.

In order to overcome the possibility of such obstacles, the present invention also considered providing additional cell types derived from ES cells. Thus, an ES cell-derived tissue modeling system has been developed. At the core of the proposed approach is parallel drug selection from a single culture of differentiating ES cells of the cell types that make up the tissue of interest. One advantage of such an approach is that the interaction between purified cell types is released from unrelated cells, using natural pathways for “crosstalk” signaling. It is processed in a “natural” way and forms a viable tissue-like structure as a product. In principle, the present invention can use many variations of such an approach:
a) Stablely transfect multiple transgenic ES clones with a specific number of vectors with drug selection cassettes triggered by specific promoters, depending on the cell types that make up the desired tissue type. In such a modification, all emerging cell types originate from the ancestor of one common ES cell clone, and the resulting ratio between individual cell components depends on their relative differentiation rate See FIG. 2A and 3B.
b) The approach using a chimeric embryoid body (EB) results in a number of transgenic ES clones, where each single clone is a drug resistance cassette initiated by one of the cell type specific promoters. Have only one vector with. For tissue modeling, the relevant clones must be mixed in the early stages of differentiation to form ES cell aggregates (EB) (“hanging drop” or “mass culture”), in this case The cell types that appear after drug selection originate from different corresponding ES cell clones, and the final ratio between the cellular components depends on and can be controlled by the initial ratio between the different ES cell lines See FIGS. 2B and 3C.

Thus, in a further aspect, the present invention provides the following steps:
(A) transfecting one or more pluripotent or multipotent cells with a recombinant nucleic acid molecule comprising first and second cell type specific regulatory sequences operably linked to at least one selectable marker. The second cell type is different from the first cell type;
(B) culturing the cell under conditions allowing differentiation of the cell;
(C) Isolating cells of at least two differentiated cell types and / or cells that have not differentiated, selectively, in the course of differentiation, independent of the target cell type that activates the selectable marker Removing together with the differentiating cells towards the different cell types;
To a method for modeling and / or obtaining a tissue or tissue-like structure.

  Further, embryonic stem (ES) cells are preferred in this method, but embryonic germ (EG) cells may be used. Similarly, the present invention relates to cells that can be differentiated into at least two cell types obtainable by the method of the present invention. Similarly, a cell aggregate composed of at least two different cell types obtainable by the method of the present invention, or a cell or tissue containing the cell aggregate obtainable by the method of the present invention is also referred to as these cell, cell aggregate. Or as well as organs, implants and grafts containing tissue.

  With the prospect of using human ES cells in tissue replacement therapy, the problem of high level purification of ES cell-derived differentiated cell types is one of the cornerstones of future ES cell-based transplantation. The high standards and reference purity required for specific cell types derived from ES cells selected for therapeutic purposes have not yet been established. To date, an approach based on drug selection of differentiated cell types derived from genetically modified ES cells in a mouse model is that there are no undifferentiated ES cells in the final product, It has been proven to be the most effective in terms of the low incidence of embryonic cancer in Japan. In addition to purity issues, the quality of transplantation of transplanted cells into recipient tissue, especially damaged tissue, is greatly influenced by supporting cells (which connect fibroblasts, stromal cells, endothelial cells, glial cells, etc.) Will depend. All of these important tissue elements are problematic in the recipient's damaged tissue and major cell types, and this creates additional problems for the process of transplanting transplanted cells, especially in their early stages . Thus, tissue modeling during human ES cell differentiation has become a modern way to obtain viable tissue prototypes with high transplantability.

Accordingly, the present invention also includes the step of implanting or transplanting cells, cell aggregates, tissues or organs obtainable by the method of the present invention into a subject in need of treatment of damaged tissue or organs. Relates to a method for treating damaged tissue or organs. In one specific aspect, the invention relates to a method for improving cardiac function in a mammal after myocardial infarction, said method comprising:
(A) a mammalian embryonic stem (ES) cell comprising a resistance gene under the control of heart, fibroblasts and optionally endothelial cell specific regulatory sequences, optionally the same specific regulatory sequences; Transfecting the recombinant nucleic acid molecule, optionally also including one or more reporters underneath;
(B) The ES cells are cultured in vitro in a medium containing a selective substance for a resistance gene under conditions that allow the ES cells to differentiate into cardiomyocytes, fibroblasts and optionally endothelial cells. Culturing; and
(C) removing undifferentiated cells from the differentiated cardiomyocytes, fibroblasts and optionally endothelial cells, optionally together with differentiating cells towards an irrelevant cell type. Steps and;
(D) aligning the differentiating cardiomyocytes, fibroblasts and optionally endothelial cells with heart-like tissue as an optional step;
(E) then co-implanting said cardiomyocytes, fibroblasts and optionally endothelial cells or said tissue into at least a portion of a previously infarcted area of heart tissue;
(F) transplanting the introduced cells or tissues in situ as viable cells located in the previously infarcted area in heart tissue, the result of the transplant being the cardiac function of the mammal Improving, including steps.

  Vectors and vector compositions comprising recombinant nucleic acid molecules as used in the methods of the invention, as well as cells containing such vectors and vector compositions, are the subject of the present invention.

  In vitro modeling of various types of tissues derived from mouse ES cells includes (i) in vitro studies on the early stages of tissue formation during embryogenesis and the effects of various types of factors and chemicals on this process. In in vitro research, it has various uses. The latter makes the proposed approach (ii) valuable for in vitro high-throughput embryo-toxicology assays, which can only test a variety of substances for their ability to influence cell type-specific differentiation. Rather, a process of “self-assembly” of differentiated cells in specialized tissue types can be demonstrated. Furthermore, the formation of such tissue-like structures in vitro obtains improved functionality and survival compared to the counterparts when alone. Thus, the method of the present invention provides (iii) a good basis for high throughput pharmacological and pharmacokinetic assays in vitro, and various compounds for which tissue targeting effects are expected. Could be tested for their direct functional effects and side effects on the tissue level. All of the above mentioned ensuing envisions a significant reduction in animal consumption that is expensive and ethical debate for both scientific and screening purposes. All of the above items relating to mouse ES cells ((i), (ii), (iii)) are fully applicable to tissue modeling from human ES cells, while at the same time they are used in embryonic studies and human models. It is emphasized that this is the only practical option available for high-throughput screening.

  In such embodiments, the use of a chip or array containing the differentiating cells of the present invention is particularly suitable. The invention therefore further relates to an array comprising a solid support and cells, cell aggregates or tissues prepared according to the invention attached to or suspended from it, in particular a microelectrode array (MEA). ) Is involved. In this context, devices adapted to analyze such arrays are also encompassed by the present invention.

Accordingly, the present invention further provides:
(A) contacting a test sample comprising cells, cell aggregates, tissues or organs prepared according to the present invention with a test substance;
(B) determining a phenotypic response in the test sample when compared to the control sample, the change in phenotypic response in the test sample when compared to the control sample is Obtaining a test substance capable of influencing cell generation and / or tissue structure formation, comprising the step of being an indicator that the test substance has an effect on cell generation and / or tissue structure formation, and / or Relates to a method for profiling.

  These methods are preferably performed on a chip or array, but are performed as appropriate in any of the methods for obtaining / modeling the tissue described herein, wherein the test sample is The test substance is contacted before, during, or after the cell or cell aggregate is subjected to the method. These screening methods can be combined with methods of manufacturing drugs, specifically methods of manufacturing drugs that support wound healing and / or healing of damaged tissue, or can be refined to use such drugs. It can also be a manufacturing method. These methods include, for example, mixing the isolated material with a pharmaceutically acceptable carrier, and packaging it in a suitable container with the corresponding formulation for the envisaged therapeutic treatment. Steps may be included.

  With respect to all the methods of the present invention, the vectors or vector compositions, arrays, pluripotent or multipotent cells that are useful for carrying out these methods and optional media, recombinants Described kits containing nucleic acid molecules, standard compounds and the like are provided.

  Other embodiments of the invention will be apparent from the description below.

DESCRIPTION OF THE INVENTION A wide variety of stem cells has become a very attractive modality in regenerative medicine. These can be grown in culture and then differentiated into cell types required for treatment in vitro or in situ. While not intending to be bound by theory, it is a feature of the present invention that some differentiated cell populations produced by adaptive culture and positive selection methods are sub-optimal for use in human therapy. It is a hypothesis. In some cases, undifferentiated cells in this population may impair cell in vivo transplantation or function. In addition, undifferentiated cells may increase the likelihood of forming malignant or other tumors at the site of therapeutic transplant or causing migration of transplanted cells. Additionally or alternatively, simply providing and transplanting certain embryonic cell types is often insufficient to achieve reconstruction of damaged tissue and the like.

  The present invention relates to protocols and methods for providing protocols for providing de novo tissues and organs that are particularly useful for transplantation and other purposes.

  In the first set of experiments in accordance with the present invention, it was possible to show that puromycin-purified cardiomyocytes show a tissue-like structure that shows integration and alignment with embryonic fibroblasts in co-culture. However, if such a tissue-like structure is comparable to or at least close to natural heart tissue, and if so, the effects observed under in vitro culture conditions can be achieved in vivo The question remained.

  In further experiments, puromycin-selected ES cell-derived cardiomyocytes can actually be successfully transplanted into the cold infarct area of the mouse heart when co-transplanted with syngeneic embryonic fibroblasts. It has been demonstrated that it can be done. These ES cell-derived cardiomyocytes exhibit various heart subtype morphology characterized by a well-developed contractile mechanism.

  Thus, a highly efficient system for drug selection and quality control of transgenic ES cell-derived cell types such as cardiomyocytes was established. The drug selection effectively increases the final yield (5 to 10 times) due to the intensive growth of cell type-specific growth, and to a level that does not take the risk of tumor development after transplantation. Reduce. Furthermore, co-culture or co-transplantation of ES cell-derived cell types with embryonic cell types belonging to connective tissues such as fibroblasts to produce natural tissues and tissue-like structures in vitro and in vivo. Can do.

  The technology of the present invention is designed, in part, to provide cell populations with superior characteristics for human therapy. After depletion of undifferentiated cells, various populations of differentiated embryonic and ES cell-derived cell types have better functional and transplant characteristics, as well as undesirable tissue structure and Expected to be less likely to cause malignancy. In addition, the development of cell populations of various embryonic and ES cell-derived cell types into tissues is more closely related to the situation in vivo. Provides a significant advantage.

Definitions For the purposes of this description, the term “stem cell” may refer to either a stem cell or a germ cell, eg, embryonic stem (ES) and germ (EG) cells, respectively. At a minimum, stem cells have the ability to proliferate to form cells of two or more different phenotypes, and self-renew when part of the same culture or when cultured under different conditions Can do. Embryonic stem cells are also typically telomerase positive and OCT-4 positive. Telomerase activity is determined using a TRAP activity assay (Kim et al., Science 266 (1997), 2011) using a commercially available kit (TRAPeze® XK telomerase detection kit, catalog s7707; Purchase, New York, Intelgen; or TeloTAGGG). (TM) telomerase PCR ELISAplus, catalog 2,013,89; Indianapolis, Roche Diagnostics). Furthermore, hTERT expression can also be evaluated by RT-PCR at the mRNA level. The LightCycler TeloTAGGG ™ hTERT quantification kit (Catalog 3,012,344; Roche Diagnostics) is commercially available for research purposes.

According to the present invention, the term embryonic stem (ES) cell is derived from pre-embryonic, embryonic, or fetal tissue at any point after fertilization and under suitable conditions, for example Multiple different origins from all three germ layers (endoderm, mesoderm, and ectoderm) according to standard tests recognized in the art, such as teratoma formation in 8-12 week old SCID mice Any pluripotent or multipotent stem cell having the characteristic that it can give rise to progeny of the cell type is encompassed.

  “Embryonic germ cells” or “EG cells” are cells derived from primordial germ cells. The term “embryonic germ cell” is used to refer to a cell of the invention that exhibits an embryonic pluripotent cell phenotype. The term “human embryonic germ cell (EG)” or “embryonic germ cell” refers to a cell of a mammal, preferably a human, of the invention that exhibits a pluripotent embryonic stem cell phenotype as defined herein. Or may be used interchangeably herein to refer to its cell line. Thus, EG cells can differentiate into cells consisting of ectoderm, endoderm, and mesoderm. EG cells can also be characterized by the presence or absence of markers associated with specific epitope sites identified by the binding of specific antibodies, or the absence of specific markers identified by the absence of specific antibody binding. .

  “Multipotent” refers to cells that retain the developmental ability to differentiate into a wide range of cell lineages, including germline. The terms “embryonic stem cell phenotype” and “embryonic stem-like cell” are also undifferentiated and thus pluripotent and can be visually distinguished from other adult cells of the same animal. It is used interchangeably here to refer to a cell.

  ES cell definition includes human embryonic stem cells as described by Thomson et al. (Science 282 (1998), 1145); rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92 (1995) 7844), marmoset stem cells (Thomson et al., Biol. Reprod. 55 (1996), 254) and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95 (1998). ), 13726) and other primate-derived embryonic stem cells. Other types of pluripotent cells are also encompassed by this term. Any cell of mammalian origin that can give rise to progeny derived from all three germ layers is encompassed, regardless of whether they are derived from embryonic tissue, fetal tissue, or other sources. The stem cells used according to the present invention are preferably (but not necessarily) preferably nucleologically normal. However, it is preferable not to use ES cells derived from malignant sources.

  “Feeder cell” or “feeder” is used to refer to a type of cell that is co-cultured with the other type of cell to provide an environment in which a second type of cell can grow. It is a term. The feeder cells are optionally derived from a different species than the supporting cells. For example, some types of ES cells are described below in this disclosure, such as primary mouse embryonic fibroblasts, immortalized mouse embryonic fibroblasts (eg, mouse STO cells such as Martin and Evans, Proc. Natl. Acad. Sci. USA 72 (1975), 1441-1445), or human fibroblast-like cells differentiated from human ES cells. The term “STO cell” refers to embryonic fibroblast mouse cells, such as those commercially available, and includes those deposited as ATCC CRL 1503.

  The term “embryoid body” (EB) is a term of art synonymous with “aggregate”. The term refers to a collection of differentiated and undifferentiated cells that appear when ES cells are overgrown in monolayer culture or maintained in suspension culture. The definitive body is typically a mixture of different cell types consisting of multiple germ layers that can be distinguished by morphological criteria. See further below.

  The terms “polynucleotide” and “nucleic acid molecule” refer to a polymer of nucleotides of any length. Genes and gene fragments, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA and RNA, nucleic acid probes and primers are included. As used in this disclosure, the term polynucleotide refers to interchangeable double-stranded and single-stranded molecules. Unless otherwise specified or sought, any embodiment of the invention that is a polynucleotide is known to form a double stranded form with a double strand, or is expected to form a double stranded form. And both of the two complementary single-stranded forms. Also included are nucleic acid analogs such as phospholeamidates and thiophospholeamidates.

  A cell may be transferred to a progeny of the first modified cell in which the polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or the cell has inherited the polynucleotide. In some cases, it is said to be “genetically modified”, “transfected”, or “genetically transformed”. Such polynucleotides will often include a transcribable sequence encoding the protein of interest that allows the cell to express the protein at a high level. A genetic modification is said to be “hereditary” if the progeny of the modified cell has the same modification.

  A “regulatory sequence” or “control sequence” is a nucleotide sequence involved in the interaction of molecules that contribute to the functional regulation of the polynucleotide, such as replication, duplexing, transcription, splicing, translation, or degradation of a polynucleotide. is there. Transcription control sequences include promoters, enhancers, and repressors.

  Certain gene sequences referred to as promoters, such as “αMHC” or “collagen” promoters, are polynucleotide sequences derived from the referenced genes that promote transcription of operably linked gene expression products. Various parts of upstream and intron untranslated gene sequences can possibly contribute to promoter activity, and all or any part of these parts can be referred to the genetically engineered constructs mentioned. It is recognized that it may exist. The promoter may be based on the gene sequence of any species having the gene, unless it says prohibition, and introduces any addition, substitution, or deletion as long as it has the ability to promote transcription in the target tissue. May be. Genetic constructs designed for human therapy typically contain segments that are at least 90% identical to the promoter sequence of the human gene. Certain sequences can be tested for activity and specificity, such as by operably linking to a reporter gene. Please refer to FIG.

Gene sequences are said to be “operably linked” when they are placed in a structural relationship that allows them to operate in a manner according to their expected function. For example, if a promoter helps to initiate transcription of the coding sequence, it can be said that the coding sequence is operably linked to (or under control of) the promoter. There may be intervening sequences between the promoter and coding region so long as this functional relationship is maintained.

  In the context of coding sequences, promoters and other gene sequences, the term “heterologous” means that the sequence is derived from an entity that is genotypically different from other parts of the entity being compared. Point to. For example, a promoter or gene introduced into a different species of animal by genetic engineering techniques is said to be a heterologous polynucleotide. An “endogenous” gene sequence is a sequence that is in the same chromosomal location as it occurs in nature, but other sequences may be artificially introduced into adjacent positions.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably in this disclosure to refer to polymers of amino acids of any length. The polymer may contain modified amino acids, may be linear or branched, and may have non-amino acids along the way.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION In one aspect, the invention comprises culturing a first cell type derived from embryonic stem (ES) cells in the presence of at least one embryonic second cell type; Integrating and aligning said at least two cell types into a tissue or tissue-like structure, to a method for modeling and / or obtaining a tissue or tissue-like structure.

  The present invention can be practiced using stem cells of any vertebrate species. Human; stem cells derived from non-human primates, livestock, livestock, and other non-human mammals are included. Among the stem cells suitable for use in the present invention are primates derived from tissues formed after pregnancy, such as blastocysts, or fetal or embryonic tissues collected at any time during pregnancy There are pluripotent stem cells. Non-limiting examples are primary cultures or established strains of embryonic stem cells. Furthermore, the present invention can be applied to adult stem cells. This is because Anderson et al., Nat. Med. 7 (2001), 393-395 and Anderson et al., 2001, Gage, FH, 200 and Prockop, Science 276 1997), 71-74.

  The medium for isolating and growing stem cells may have any of a number of different formats, so long as the resulting cells have the desired characteristics and are capable of growing. Suitable sources include Iscove's modified Dulbecco medium (IMDM), Gibco, # 12440-053; Dulbecco's modified Eagle medium (DMEM), Gibco # 11965-092; Knockout Dulbecco's modified Eagle medium (KO DMEM) , Gibco # 10829-018; 200 mM L-glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; [beta] -mercaptoethanol, Sigma # M7522; human recombinant basic fiber There is blast growth factor (bFGF), Gibco # 13256-029. Exemplary serum-containing ES media and conditions for culturing stem cells are known and can be optimized as appropriate depending on the cell type. Media and culture techniques for the specific cell types mentioned in the previous section are provided in the references cited herein.

  As previously mentioned, several sources for ES cells can be arbitrarily selected by those skilled in the art, among which human stem cells are the majority of embodiments of the invention, particularly for therapeutic purposes such as transplantation. For this, it is preferable. Human embryonic stem cells and their use to prepare various cell and tissue types are also described in Reprod. Biomed. Online 4 (2002), 58-63. Embryonic stem cells can be isolated from blastocysts of primate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92 (1995), 7844). Human embryonic germ (EG) cells can be prepared from primordial germ cells present in human fetal material collected about 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95 (1998), 13726. A method for producing embryonic stem cells or cells resembling embryonic germ cells in terms of morphology and pluripotency derived from primordial germ cells isolated from human embryonic tissues, such as from the gonads of human embryos, U.S. Pat. No. 6,245,566.

  Recently, human deciduous deciduous teeth, a tissue that is relatively easy to obtain, is a highly proliferative clonogenic cell population that can differentiate into a variety of cell types including neurons, adipocytes, and tooth-forming cells. It was reported to contain pluripotent stem cells that were identified. See Miura et al., Proc. Natl. Acad. Sci. USA 100 (2003), 5807-5812. After in vivo transplantation, these cells were found to induce bone formation, produce dentin, and survive in the mouse brain with the expression of neuronal markers. Furthermore, Lin et al. In Stem Cells 21 (2003), 152-161 explained the multilineage of homozygous stem cells derived from metaphase II oocytes. A variety of sources of progenitor cells of the muscle immediately after birth and factors that may enhance stem cell involvement in the formation of new skeletons and myocardium in vivo are grounds et al., J. Histochem. Cytochem. 50 (2002), 589-610. A method for purifying the few hematopoietic stem cells (HSC) returning to the bone marrow to homogeneity is described in US application US2003 / 0032185. It has been described that these adult bone marrow cells have enormous differentiation potential so that they can also differentiate into liver, lung, gastrointestinal tract and skin epithelial cells. This discovery will contribute to the clinical treatment of genetic diseases or tissue repair. In addition, techniques such as nuclear transfer for embryo reconstruction may be utilized, in which case diploid donor nuclei are transplanted into enucleated MII oocytes. This technique, combined with other techniques that help establish a custom-made, embryonic stem (ES) cell line that is genetically identical to that of the recipient, is the Colman and Kind, Trends Biotechnol. 18 (2000), 192- Reviewed in 196 In order to avoid graft rejection associated with allogeneic or xenogeneic cells in transplantation, it is preferred to use syngeneic or autologous cells and recipients in corresponding embodiments of the invention. Given the recently discovered stem cell sources, such as bone marrow and dental origin, it should be possible to meet this requirement without having to rely on embryonic cells and tissues. Alternatively, the cells may be genetically manipulated to suppress the associated transplant antigen. See also below. An immunosuppressant may be used.

  The field of stem cell technology is reviewed by Kiessling and Anderson, Harvard Medical School, in human Embryonic Stem Cells: An Introduction to the Science and Therapeutic Potential; (2003) Jones and Bartlett Publishers; ISBN: 076372341X.

  The use of human embryos or the like as stem cell donors seems to be justifiable at least under certain circumstances, but in order to avoid this, non-human transgenic animals, especially mammals, are the source of embryonic stem cells. It would also be possible to use For example, compositions and methods for making transgenic pigs for use as xenograft donors are described in US Pat. No. 5,523,226. Similarly, international application WO97 / 12035 describes a method for producing transgenic animals for xenotransplantation. Furthermore, an immunologically compatible animal tissue suitable for xenotransplantation into human patients is described in international application WO01 / 88096. Methods for producing embryonic germ cells from pigs are described, for example, in US Pat. No. 6,545,199.

  Stem cells can be grown continuously in culture using a combination of culture conditions that promote proliferation without promoting differentiation. Classically, stem cells are cultured on a layer of feeder cells, typically fibroblast type cells, often derived from embryonic or fetal tissue. This cell line is plated to near confluence and is usually irradiated to prevent growth and then by certain cells (eg Koopman and Cotton, Exp. Cell 154 (1984), 233-242; Smith and Hooper, Biol. 121 (1987), 1-91) or used in support when cultured in medium conditioned by exogenous addition of leukemia inhibitory factor (LIF). Such cells can be grown indefinitely using suitable culture conditions.

  International applications WO03 / 010303 and Mummery et al., Circulation 107 (2003), 2733-2740 disclose experiments using human embryonic stem (hES) cells that differentiate into cardiomyocytes. The hES cells were co-cultured with mouse-derived visceral-endoderm (VE) -like cells. In these experiments, mouse endoderm cells replace the commonly used mouse fibroblast feeder cells and are used to induce cardiomyocyte differentiation in hES cells that do not undergo spontaneous cardiac development .

  Thus, Mummery et al. Do not teach a method of providing a tissue or tissue-like structure that allows the endoderm cells to integrate and align with hES cells. Conversely, the use of mouse endoderm cells has already been shown to be removed when using differentiated muscle cells for further uses, including transplantation. Furthermore, in contrast, the methods of the present invention typically utilize stem and embryonic cells generated from the same species, most preferably a human.

  In the absence of feeder cells, exogenous leukemia inhibitory factor (LIF), or conditioned medium, ES or EG cells will spontaneously differentiate into cells found in endoderm, mesoderm, and ectoderm. A wide variety of cell types including. However, cell differentiation can be controlled by appropriately combining growth factors and differentiation factors. For example, mouse ES and EG cells can generate hematopoietic cells in vitro (Keller et al., Mol. Cell Biol. 13 (1993), 473-486; Palacios et al., Proc. Natl. Acad. Sci. USA 92 (1995), 7530-7534; Rich, Blood 86 (1995), 463-472). In addition, mouse ES cells are neurons (Bain et al., Developmental Biology 168 (1995), 342-357; Fraichard et al., J. Cell Science 108 (1995), 3161-3188), cardiomyocytes (heart Muscle cells) (Klug et al., Am. J. Physiol. 269 (1995), H1913-H1921), skeletal muscle cells (Rohwedel et al., Dev. Biol. 164 (1994), 87-101), blood vessels It has been used to generate in vitro cultures of cells (Wang et al., Development 114 (1992), 303-316). US Pat. No. 5,773,255 relates to a glucose responsive insulin secreting pancreatic beta cell line and US Pat. No. 5,789,246 relates to hepatocyte progenitor cells. Liver differentiation of mouse embryonic stem cells is also described in Jones et al., Exp. Cell Res. 272 (2002), 15-22.

  Other progenitor cells involved include, but are not limited to, chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal canal epithelial cells, There are smooth and skeletal muscle cells, testicular progenitor cells, and vascular endothelial cells. An embryonic stem cell differentiation model for cardiac development, myogenesis, neurogenesis, epithelial and vascular smooth muscle differentiation in vitro is outlined in Guan et al., Cytotechnology 30 (1999), 211-226. Yes.

  In some embodiments of the invention, differentiation is promoted by collecting one or more media components that promote the growth of undifferentiated cells or act as inhibitors of differentiation. Examples of such components are several growth factors, mitogens, leukocyte inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). Furthermore, differentiation may also be promoted by adding media components that promote differentiation towards the desired cell lineage or inhibit the growth of cells with undesirable characteristics.

  According to the present invention, a selection system that is lethal to undesired cells and cell types is used under the control of regulatory sequences that preferentially express the gene in the desired cell types and / or at specific developmental stages. That is, depleting relatively undifferentiated cells from a differentiated cell population by expressing a selectable marker gene that renders cells of a particular cell type resistant to the lethal effects of external agents. And / or depleting cells of undesired cell types. To accomplish this, prior to the process used to differentiate the cell into the desired lineage for treatment, the cell is genetically modified to a first cell type specific for the desired first cell type. It includes a selectable marker operatively linked to a specific regulatory sequence. An example of the construct is given in FIG.

  Any suitable expression vector can be used for this purpose. Suitable viral vector systems for producing stem cells modified according to the present invention can be prepared using commercially available viral components. The introduction of one or more vector constructs into embryonic stem cells is carried out in a known manner, for example with the help of transfection, electroporation, lipofection or viral vectors. Viral vectors containing effector genes are outlined in the published literature mentioned in the last section. Alternatively, vector plasmids can be introduced into cells by electroporation or by using lipid / DNA complexes. An example is the formulation Lipofectamine 2000 (TM) available from Gibco / Life Technologies. Another example of a reagent is FuGENE (TM) 6 Transfection Reagent, a mixture of non-liposomal lipids and other compounds in 80% ethanol, available from Roche Diagnostics. . Preferably, the vector construct and transfection method described in International Application WO02 / 051987, the disclosure of which is incorporated herein by reference, may be used.

  The resistance gene itself is known. Examples of these are nucleoside and aminoglycoside-antibiotic resistance genes for eg puromycin (puromycin-N-acetyltransferase), streptomycin, neomycin, gentamicin or hygromycin. Further examples of resistance genes are multidrug resistance genes that confer resistance to a number of antibiotics, such as dehydrofolate-reductase that confer resistance to aminopterin and methotrexate, and vinblastine, doxorubicin and actinomycin D, for example.

  In a particularly preferred embodiment of the invention, the selectable marker provides resistance to puromycin. Puromycin is particularly suitable for rapidly removing cells other than heart cells in transgenic cultures of transgenic EBs. Furthermore, drug selection for cardiac cells can be performed completely in suspension culture of transgenic EBs. Thus, it was also possible to show that purified ES cell-derived cardiomyocytes survive longer in culture than their untreated counterparts. Furthermore, removal of undifferentiated ES cells during the drug selection process alone has been shown to have a distinct positive effect on the viability and longevity of such differentiated ES cell-derived cells as cardiomyocytes. . In addition, it was surprisingly possible to show that release from surrounding undifferentiated cells induced cardiomyocyte proliferation. Thus, drug selection has an effect on both purification and growth.

  In a preferred embodiment of the present invention, the ES cell of the first cell type derived from the ES cell contains a reporter gene, wherein the reporter is a cell type-specific regulatory sequence specific to the first cell type. Is operatively connected to. This type of vector has the advantage of providing differentiation visualization, defining the start of drug selection, visualizing drug selection, and tracking the fate of purified cells transplanted into recipient tissue. Such vectors that are preferably utilized in accordance with the method of the present invention are described in International Application WO02 / 051987. Usually, the cell type specific regulatory sequence of the reporter gene is substantially the same as the first cell type specific regulatory sequence of the marker gene. This involves placing the marker gene and the reporter gene in the same recombinant nucleic acid molecule, i.e., a vector used for stem cell transfection, preferably such that the marker gene and the reporter gene are contained on the same cistron. This can also be achieved advantageously. An example of a bicistronic heart-specific drug selection cassette-reporter vector is shown in FIG.

  The reporter may be of any kind as long as it does not damage the cells and provides an observable or measurable phenotype. According to the present invention, green fluorescent protein (GFP) and its derivatives from the jellyfish equorea-victoria (described in international applications WO95 / 07463, WO96 / 27675 and WO95 / 121191) Blue GFP "(Heim et al., Curr. Biol. 6 (1996), 178-182 and" Redshift GFP "(Muldoon et al., Biotechniques 22 (1997), 162-167) can be used. What is enhanced green fluorescent protein (EGFP) Further embodiments are enhanced yellow and cyan fluorescent proteins (EYFP and ECFP, respectively) and red fluorescent proteins (DsRed, HcRed), additional fluorescent proteins known to those skilled in the art. As long as the cells are not damaged, the fluorescent protein can be detected through a fluorescent detection method known per se, for example, Kolossov et al., J. Cell Biol. 143 (1998), See 2045-2056 Fluorescence Instead of proteins, especially for in vivo applications, other detectable proteins can be used, especially epitopes of these proteins, proteins that may themselves damage the cell itself, if the epitope is a cell The epitope can be used as long as it is not damaged, see also international application WO02 / 051987.

For selection of stably transfected ES cells, the vector construct contains an additional selection marker gene that provides resistance to antibiotics such as neomycin. Of course, other known resistance genes can also be used, such as the resistance genes described above in connection with fluorescent protein coding genes. The selection gene for selection of stably transfected ES cells is under the control of a promoter that is different from that which controls the control of detectable protein expression. Often, a constitutively active promoter is used, such as a PGK-promoter.

Using a second selection gene is advantageous with respect to the ability to identify clones that were successfully transfected in the first place (with relatively low efficiency). Otherwise, there may be a vague majority of untransfected ES cells, and no EGFP positive cells are detected during differentiation.

  In a further embodiment of the invention, the cells can be additionally manipulated so that no specific tissue is formed. This can be done, for example, by inserting a repressor sequence such as a doxycycline inducible repressor sequence. Thereby, the possibility of contaminating the desired differentiated cells with pluripotent and potentially tumorigenic cells can be eliminated.

  The desired first cell type intended for the stem cell to be differentiated may be of any type, including but not limited to neurons, glial cells, cardiomyocytes, glucose-responsive insulin secreting pancreas Beta cells, hepatocytes, astrocytes, oligodendrocytes, chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts, keratinocytes, dendritic cells, hair follicle cells, renal canal epithelial cells, vascular endothelial cells, testicular progenitors There are cells, smooth muscle and skeletal muscle cells. See also above.

  In a specific preferred embodiment of the invention, the first cell type is a cardiomyocyte. In this embodiment, the first cell type specific regulatory sequence is preferably atrial and / or ventricular specific. Corresponding regulatory sequences, ie heart-specific promoters, are specific for very early cardiomyocytes and mesoderm progenitors, respectively, Nkx-2.5 (Lints et al., Development 119 (1993), 419-431); Specific human heart-α-actin (Sartorelli et al., Genes Dev. 4 (1990), 1811-1822), and MLC-2V specific for ventricular cardiomyocytes (O'Brien et al., Proc. Natl. Acad. Sci. USA 90 (1993), 5157-5161 and international application WO96 / 16163). The heart-specific alpha-myosin heavy chain promoter is Palermo et al., Cell Mol. Biol. Res. 41 (1995), 501-519; Gulick et al., J. Biol. Chem. 266 (1991), 9180-91855 The light chain-2v (MLC2v) promoter of this myosin is Lee et al., Mol. Cell Biol. 14 (1994), 1220-1229; Franz et al., Circ. Res. 73 (1993) , 629-638. See also the expression of atrial specific myosin heavy chain AMHC1 as described in Yutzey et al., Development 120 (1994), 871-883, and the establishment of anteroposterior orientation in the developing chicken heart.

  Mueller et al., Transcriptional Control of Enhanced Green Fluorescent Protein (EGFP) with a Cytomegalovirus (CMV (enh)) Ventricular Specific 2.1 kb Myosin Light Chain 2v (MLC-2v) Promoter and a 0.5 kb Enhancer Sequence Describes the selection of ES cell-derived ventricular-like cardiomyocytes in vitro by using below. See Mueller et al., FASEB J. 14 (2000), 2540-2548. This publication also describes the electrophysiological studies that could be similarly performed on the in vitro generated tissues and tissue-like structures of the present invention.

  In particular, based on embodiments relating to in vitro differentiated cardiomyocytes, it is preferred to use fibroblasts as the at least one embryonic second cell type. As shown in the examples, when ES cell-derived cardiomyocytes and embryonic fibroblasts were co-cultured and co-transplanted, respectively, heart tissue formation and a successful replacement therapy were achieved. These fibroblasts do not necessarily have to be derived from embryos, and can be generated de novo from ES cells according to the method of the present invention. Thus, ES cells are operably linked to markers and optionally cell type-specific regulatory sequences, i.e. fibroblast-specific promoters such as a2 (I) collagen promoter that are also active in bone cells. Recombinant nucleic acid molecules containing the generated reporter gene (Lindahl et al., J. Biol. Chem. 277 (2002), 6153-6161; Zheng et al., Am. J. Pathol. 160 (2002 ), 1609-1617; Antoniv et al., J. Biol. Chem. 276 (2001), 21754-21764; further Finer et al., J. Biol. Chem. 262 (1987), 13323-13333; Bou-Gharios et al., J. Cell Biol. 134 (1996), 1333-1344; Zheng et al., Am. J. Pathol. 160 (2002), 1609-1617; Metsaranta et al., J. Biol. Chem. 266 (See also (1991) 16862-16869).

  However, in other embodiments, fibroblasts and / or alternatively other supporting cells such as, for example, endothelial cells and their derivatives may be used.

  In a further preferred embodiment, the method of the invention further comprises culturing said at least two cell types in the presence of a third cell type derived from embryonic or embryonic stem (ES) cells. The third cell type may be any of the cell types described above. Preferably, the third cell type is an endothelial cell. Therefore, either embryonic endothelial cells or ES cell-derived endothelial cells may be used. In the latter embodiment, the endothelial cells are derived from ES cells transfected with a vector construct as outlined generally above, in which case the cell type specific regulatory sequence is endothelial cell specific. Promoter. For example, vascular endothelial cell-cadherin promoter as described by Gory et al., Blood 93 (1999), 184-192; according to Schlaeger et al., Proc. Natl. Acad. Sci. USA 94 (1997), 3058-3063 See Tie-2 promoter / enhancer; and Flk-1 promoter / enhancer according to Kappel et al., Biochem. Biophys. Res. Commun. 276 (2000), 1089-1099, etc.

  Additional cell and tissue type specific promoters are known. For example, a chondrocyte-specific pro-alpha1 (II) collagen chain (collagen 2) promoter fragment as described in Zhou et al., J. Cell Sci. 108 (1995), 3677-3684; Gloster et al., J. Neurosci. 14 (1994); 7319-7330, a neuronal alpha-1-tubulin specific promoter; and Besnard et al., J. Biol. Chem. 266 (1991), 18877-18883. See acidic protein (GFAP) promoter. Further examples of tissue-specific promoters are those that are active in glial cells, hematopoietic cells, neurons, preferably embryonic neuronal cells, endothelial cells, chondrocytes or epithelial cells or insulin secreting β cells. “Tissue specific” is encompassed by the term “cell specific”.

  Further examples of non-cardiac specific promoters are PECAM1, FLK-1 (endothelium), nestin (neural progenitor cells), tyrosine-hydroxylase-1-promoter (dopaminergic neurons), smooth muscle α-actin, smooth muscle Myosin (smooth muscle), α1-fetoprotein (endothelium), smooth muscle heavy chain (SMHC minimal promoter (specific for smooth muscle, Kallmeier et al., J. Biol. Chem. 270 (1995), 30949-30957) is there.

  The term development-specific promoter refers to a promoter that is active at a specific time during development. Examples of such promoters are β-MHC promoters that are expressed during embryonic development in the mouse ventricle and replaced by α-MHC promoters in the prenatal stage, early mesoderm / heart promoters NKx2.5, with the exception of regulators that are down-regulated in late developmental stages, atrial natriuretic factor, a marker of early embryonic heart, Flk-1, an endothelium-specific promoter active during early angiogenesis, neural progenitor Intron 2-segment of nestin gene expressed in cells (embryonic neurons and glial cells) and adult glial cells (partially still capable of division) (Lothian and Lendahl, Eur. J. Neurosci. 9 (1997) 452-462U).

  In the above embodiment, the vectors shown in FIGS. 1 to 3 are preferably used.

  The invention further relates to cell co-cultures defined by the methods described herein, and tissues obtainable by the methods of the invention. Cells and tissues prepared according to the present invention can be used for a variety of commercially important research, diagnostic and therapeutic purposes. Since the cell population of the present invention is depleted of undifferentiated cells, these can be used to prepare antibodies and cDNA libraries specific for the phenotype after differentiation. General techniques used in generating, purifying, and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in the Handbook of Experimental Immunology (Weir & Blackwell, eds.); Current Protocols in Immunology (Coligan et al., eds.); and Methods of Immunological Analysis (Masseyeff et al., eds., Weinheim: VCH Verlags GmbH). General techniques involved in preparing mRNA and cDNA libraries are: RNA Methodologies: A Laboratory Guide for Isolation and Characterization (RE Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000).

However, one of the main objects of the present invention is to provide cells and tissues for use in transplantation. For example, the differentiated cells of the present invention can be used for this in human patients in need of tissue remodeling or regeneration. The cells are administered in such a way that they can be transplanted into the tissue site of interest to reconstruct or regenerate a functionally defective area. Thus, the present invention specifically includes:
(A) introducing a cell inoculum or corresponding tissue that has been induced to differentiate, preferably comprising a co-culture of transgenic stem cells, into at least a portion of the tissue previously damaged; ,
(B) transplanting the introduced cellular inoculum in situ as viable cells or tissues placed in a previously damaged area of the tissue, the result of the transplantation, Improving the tissue repair and / or organ function in a mammal, comprising the step of improving the mammalian tissue and / or organ function.

  The potential of stem cells using examples selected both in terms of their ability to differentiate into specific cell types and their ability to treat a variety of diseases and conditions such as Parkinson's disease, spinal cord injury, diabetes, and heart disease A description of the potential is reviewed in Pfendler and Kawase in Obstet. Gynecol. Surv. 58 (2003), 197-208. All these conditions can be treated by the use of the cells and tissues described above.

  In one specific aspect, the present invention relates to a method for significantly improving cardiac function and repairing cardiac tissue in a living mammalian subject after the occurrence of myocardial infarction or tissue damage. The method introduces and transplants embryonic stem cells, ie, mammalian embryonic stem cell-derived cardiomyocytes, together with supporting embryonic cells such as embryonic fibroblasts, into an infarcted or damaged area of the myocardium. Surgical technique. After transplantation, the cells form a stable transplant and survive indefinitely within the infarcted or damaged area of the heart in a living host. The proven beneficial effects of the present invention include a reduction in infarct area and an improvement in cardiac function. See FIG.

Accordingly, the present invention further relates to a method for improving cardiac function in a mammal after myocardial infarction, said method comprising:
(A) Conditions under which an undifferentiated mammalian embryonic stem (ES) cell containing a resistance gene and a reporter gene under the control of the same heart-specific promoter can be differentiated into cardiomyocytes in vitro. And culturing in a medium containing a selective substance for said resistance gene,
(B) The differentiated cardiomyocytes are isolated and / or cells that have not differentiated are optionally removed together with the differentiating cells towards a cell type unrelated to the cardiomyocytes in the course of differentiation And steps to
(C) then co-implanting the cardiomyocytes together with embryonic or ES cell-derived fibroblasts and / or endothelial cells into at least a portion of a previously infarcted area of heart tissue;
(D) transplanting the introduced cellular inoculum in situ as viable cells placed in a previously infarcted area in the heart tissue, wherein the transplantation results in the mammalian heart The function is improved.

  Similar to the embodiment described here, the resistance gene and the reporter gene are contained in a bicistronic vector, but are preferably separated by IRES. Particularly preferred is the use of a construct in which the resistance gene confers resistance to puromycin, the marker is EGFP and the promoter is a cardiac αMHC promoter. Please refer to FIG. Transplantation of differentiation-induced embryonic stem cells and determination of cardiac function can be performed as disclosed in Examples and References, or US Pat. No. 6,534,052.

  US Pat. No. 5,733,727 describes myocardial transplants of skeletal myoblasts or cardiomyocytes, and cellular compositions and methods useful in obtaining such transplants. These myocardial transplants are described as being stable and suitable for use, such as for delivering recombinant proteins directly to the heart. This US patent only describes the common approach to making ES cell-derived cardiomyocytes and their use at the time of implantation and as a delivery vehicle for delivery of recombinant proteins to the heart. It may be applicable to tissues and tissue-like structures obtained according to the invention. Thus, specifically, the in vitro-generated heart tissue-like structure of the present invention is converted into angiogenic factors (for example, basic and acidic fibroblast growth factors, transforming growth) for inducing angiogenesis. Factor-β, vascular endothelial growth factor and hepatocyte growth factor). Similarly, transplants that express neurotrophic substances in the vicinity of the infarct region may be used to mitigate the occurrence of arrhythmias associated with the border zone. These and many other candidate substances for targeted delivery to the heart will be apparent to those skilled in the art.

As described above, according to the present invention, any of the at least two cell types such as main cell types and the corresponding supporting cells may be derived from ES cells. Accordingly, in a further aspect, the present invention provides the following steps:
(A) transfecting one or more pluripotent or multipotent cells with a recombinant nucleic acid molecule comprising first and second cell type specific regulatory sequences operably linked to at least one selectable marker. The second cell type is different from the first cell type; and
(B) culturing the cells under conditions allowing differentiation of the cells;
(C) Isolating cells of at least two differentiated cell types and / or cells that have not differentiated, optionally cells unrelated to the target cell type that activates the selectable marker during differentiation Removing together with differentiating cells towards the species;
To a method for modeling and / or obtaining a tissue or tissue-like structure.

  Similar to the method described above, preferably more than two cell types are generated. Thus, the method preferably transfects said one or more cells with a recombinant nucleic acid molecule comprising at least one additional cell type specific regulatory sequence operably linked to at least one selectable marker. A step, wherein the at least one additional cell type is different from the first and second cell types. When used in this method, the recombinant nucleic acid molecules are contained within the same vector or different vectors. The principles underlying these choices are shown in FIGS. 2 and 3 and described in the examples.

  The cell types are neurons, glial cells, cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes, chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts, keratinocytes , Dendritic cells, hair follicle cells, renal canal epithelial cells, vascular endothelial cells, testicular progenitor cells, smooth muscle and skeletal muscle cells. See also above.

  Preferred promoters for the preparation of heart tissue by differentiating the transfected stem cells into cardiomyocytes, fibroblasts and optionally endothelial cells include those previously described. Similarly, when generating neural tissue, one or more stem cells, such as pluripotent neural stem cells, can be genetically manipulated according to the present invention to differentiate into neurons, astrocytes, and oligodendrocytes. The same principle applies to the production of liver or pancreatic tissue. The regulatory sequences of the corresponding cell type specific promoter can be obtained from the literature. See for example “medline” and NCBI.

  It should be understood that when performing the methods of the invention, the one or more recombinant nucleic acid molecules can be simultaneously or sequentially transfected into the one or more cells.

  As described in the examples and shown in FIGS. 2 and 3, the method of the present invention can be performed in various ways. First, preferably as described herein, a multiplex stably transfected with a specific number of vectors with drug selection cassettes that are operated by specific promoters based on the cell types that make up the desired tissue type. Transgenic ES cell clones are created. Thus, at least one of the ES cells or cell clones thereof is transfected and selected, wherein the cells or cell clones are recombinant with at least two different cell type specific regulatory sequences. Contains nucleic acid molecules. In such variants, all emerging cell types originate from a common primordial ES cell clone, and the resulting ratio between the various cellular components depends on their relative differentiation rate .

  Alternatively, at least two different ES cells or clones thereof are transfected and selected, in which case the at least two different cells or cell clones are recombinant nucleic acids having different cell type specific regulatory sequences. Contains molecules. This approach results in a number of transgenic ES cell clones, where each single clone has only one vector with a drug resistance cassette that is operated by one of the cell type specific promoters.

  In the case of tissue modeling, in order to form an ES cell aggregate (EB), the relevant clones must be mixed at an early stage of differentiation (“hanging drop” or “bulk culture”), in which case The cell types that appear after drug selection originate from various corresponding ES cell clones, and the final ratio between the cellular components depends on the initial ratio between the various ES cell lines and can be controlled by this initial ratio. is there. As a result of this method, it is preferred that a cell aggregate that is a chimeric embryoid body (EB) is produced.

  Regardless of the specific embodiment of the method of the invention, it is preferred that at least two of the selectable markers operably linked to the different cell type specific regulatory sequences are identical. As mentioned above, one or more of these marker genes are preferably selectable markers that confer resistance to cytotoxic agents, preferably puromycin, methotrexate, or neomycin.

  As already discussed with respect to the method of the first aspect of the invention, the one or more of the recombinant nucleic acid molecules preferably further comprise a reporter operably linked to the cell type specific sequence. Good. See above. Also suitable here are different colored forms of enhanced green fluorescent protein (EGFP) operatively linked to different cell type specific sequences, specifically EYFP (yellow), ECFP (blue) and / or hcRFP (Red). Similarly, it is also preferred that the selection marker and the reporter are expressed from a bicistronic vector, wherein the selection marker and the reporter are one or more internal operably linked to at least one of the genes. It is preferred that they are separated at the ribosome entry site (IRES).

  As described above, the method of the present invention is preferably performed so that different cell types can be self-assembled into, for example, a desired tissue or tissue-like structure. In a preferred embodiment of the present invention, the stem cells are obtained in the form of aggregates known as embryoid bodies. International application WO02 / 051987 describes a protocol for obtaining embryoid bodies. The production is preferably carried out by the “hanging drop” method or by methylcellulose culture (Wobus et al., Differentiation 48 (1991), 172-182).

  Instead of this, a stirring flask (stirring culture) can be used as a culture method. Therefore, undifferentiated ES cells are introduced into a stirred culture and mixed irreversibly according to established techniques. For example, 10 million ES cells are introduced into 150 ml medium supplemented with 20% FCS and stirred constantly at a speed of 20 rpm. In this case, the direction of stirring motion is regularly changed. 24 hours after the introduction of ES cells, an additional 100 ml of medium supplemented with serum is added, and then 100-150 ml of medium is changed daily (Wartenberg et al., FASEB J. 15 (2001), 995). -1005). Under these culture conditions, a large amount of ES cell-derived cells, that is, cardiomyocytes, endothelial cells, neurons, etc. can be obtained according to the composition of the medium. Cells are selected using resistance genes, either still in agitation culture or after plate, respectively.

  Alternatively, EBs differentiated by hanging drop may be simply left in suspension without being plated. Even under these conditions, the progress of differentiation could be observed empirically. Undesired cell types can be washed away by mechanical mixing alone or by adding a low concentration of enzyme (eg, collagenase, trypsin). Single cell suspension is achieved by simple washing of undesired cell types.

  In certain embodiments, the fate of a cell type, the formation of cell aggregates and tissues, and the physiological and / or developmental status of the cells or cell aggregates, such as isometric tensile measurements, echocardiography, etc. Analyze by. Preferably, the status of the cells or cell aggregates may be analyzed by observing the differentiation of the electrical activity of the cells on the array, for example by recording the extracellular field potential with a microelectrode array (MEA). For example, tracking the electrophysiological characteristics of embryonic stem cells that differentiate into cardiomyocytes during the process of differentiation by recording the external field potential with a microelectrode array consisting of 60 substrate-integrated electrodes. Can do. See Banach et al. Am. J. Physiol. Heart Circ. Physiol. 284 (2003), H2114-H2123. Using multiple arrays of tungsten microelectrodes, simultaneous responses of intracerebral stem neurons contributing to respiratory movement pattern generation were recorded. See Morris et al., Respir. Physiol. 121 (2000), 119-133.

  The present invention further relates to cells, cell aggregates and tissues obtainable by the above-described method, wherein the cells can be differentiated into at least two cell types. Thus, the cells are preferably embryonic cell types--and / or tissue specific cells, most preferably heart tissue. Similarly, organs consisting of these cells, cell aggregates and tissues are the subject of the present invention, as are implants or transplants comprising such cells, cell aggregates, tissues or organs. All of these can be used in a method of treating a damaged tissue or organ of a subject, including the step of implanting or transplanting into the subject in need of treatment of the damaged tissue or organ. Accordingly, compositions such as pharmaceutical compositions comprising any one of the recombinant nucleic acid molecules, cells, cell aggregates, or tissues of the invention as described herein are within the scope of the invention. As mentioned above, these compositions and methods of the present invention are used for a variety of purposes, such as analyzing the early stages of tissue formation during embryonic development or analyzing the effects of factors and compounds on this process. be able to.

  In a further embodiment, the present invention relates to non-human transgenic animals that can be made from the mentioned ES cells and ES cell-derived cell types and cell aggregates. See above. The production of transgenic animals from ES cells is known in the art. See, for example, A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. General methods for generating non-human transgenic animals have been described in the art, see eg international application WO94 / 24274.

In one particularly preferred aspect, the present invention relates to a method for improving cardiac function in a mammal after myocardial infarction, said method comprising:
(A) a set comprising a resistance gene under the control of heart, fibroblast and optionally endothelial cell specific regulatory sequences, and optionally including one or more reporters under the same specific regulatory sequence Transfecting a recombinant nucleic acid molecule into a mammalian embryonic stem (ES) cell;
(B) in a medium containing a substance selective for the resistance gene under conditions allowing differentiation of the ES cells into cardiomyocytes, fibroblasts and optionally endothelial cells; culturing in vitro;
(C) removing undifferentiated cells from the differentiated cardiomyocytes, fibroblasts and optionally endothelial cells, optionally together with differentiating cells towards irrelevant cell types;
(D) integrating and aligning the differentiating cardiomyocytes, fibroblasts and optionally endothelial cells with heart-like tissue as an optional step;
(E) then co-implanting the cardiomyocytes, fibroblasts and optionally endothelial cells or the tissue into at least a portion of the heart tissue previously infarcted;
(F) transplanting the introduced cells or tissues in situ as viable cells placed in a previously infarcted area of the heart tissue, wherein the transplantation results in the mammalian Improving the cardiac function.

  As mentioned above, the cardiomyocytes, fibroblasts and optionally endothelial cells are preferably derived from the same ES cell. However, cardiomyocytes, fibroblasts and optionally endothelial cells derived from different ES cells may also be used. In these embodiments, the cardiac specific regulatory sequence is preferably selected from the promoters of αMHC, MLC2v, MLC1a, MLC2a and beta MHC, and the endothelial cell specific regulatory sequence is preferably Tie2, The Tie1 and caterin promoter may be selected, and the fibroblast-specific regulatory sequence is preferably selected from the collagen I promoter. See above. Similarly, the cardiomyocytes, fibroblasts and optionally the reporters of endothelial cells are preferably individually selected from enhanced green fluorescent protein ECFP (blue), EYFP (yellow) and hcRFP (red). See also FIG. 3 and the examples. The resistance gene and the reporter are preferably separated by an internal ribosome entry site (IRES).

  In another example, neuroepithelial cells are generated and used to augment or replace cells damaged by disease, autoimmune abnormalities, accidental damage, or genetic abnormalities. In vitro differentiation of mouse ES cells with retinoic acid to form neuronal progenitor cells and glial progenitor cells that are positive for astrocyte (GFAP) or oligodendrocyte (O4) markers, and then functional neurons (Fraichard et al., J. Cell Science 108 (1995), 3161-3188). It has been observed that cells transplanted into the adult brain innervate the host striatum (Deacon et al., Exp. Neurology, 149 (1998), 28-41). Human and mouse EC cell lines can also differentiate into neurons. (Trojanowski et al., Exp. Neurology, 144 (1997), 92-97; Wojcik et al., Proc. Natl. Acad. Sci. USA, 90 (1993), 1305-1309). When these neurons were transplanted into rats with cerebral ischemia, the degree of functional recovery was enhanced (Borlongan et al., Exp. Neurology 149 (1998), 310-321). According to the present invention, corresponding neuron and glial specific promoters are used for this embodiment. See, for example, Kawai et al., Biochim. Biophys. Acta 1625 (2003), 246-252, and Kugler et al., Gene Ther. 10 (2003), 337-347 for glial and neuron specific promoters. . The efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in various culture systems is described, for example, in Dang et al., Biotechnol. Bioeng. 78 (2002), 442-453. In another application of the invention, ES cells or their differentiating or post-differentiation derivatives can be used to make non-cellular structures such as bone or cartilage replacements. In another application of the invention, ES cells or their differentiation during or after differentiation can be used for the production of liver tissue. Regulatory sequences for cell type specific expression can be obtained from cited sources and general sources such as “medline” and NCBI.

  If desired, such cells may be genetically modified for gene therapy purposes.

  In a further aspect, the present invention relates to a vector or vector composition comprising a recombinant nucleic acid molecule as defined in the context of the inventive method described above. Specifically, the present invention comprises a total of at least two units of resistance genes under the control of heart, fibroblast and optionally endothelial cell specific regulatory sequences, and the same specificity as described above Relates to vectors and vector compositions, optionally comprising one or more reporters underneath the regulatory sequences. See further FIG. 3A. These vectors or vector compositions may be substantially isolated or may be present in the sample, such as in one or more useful host cells, eg, vector propagation.

  In one particularly preferred embodiment, the present invention comprises a solid support and a cell obtained by the method of the present invention or in the process of differentiation, attached to or suspended from said support. And an array comprising an aggregate or tissue. Of particular interest is the use of planar microelectrode arrays as bioarrays for cultured cells and cell aggregates. Such arrays generally consist of a glass, plastic or silicon substrate and a conductor such as gold, platinum, iodine-tin oxide or iridium deposited or patterned on the substrate. For example, an insulating layer such as photoresist, polyimide, silicon dioxide, or silicon nitride is deposited on the conductive electrode, interconnects the regions on the electrode, and then removed to define the recording site. The cells are cultured directly on this surface and come into contact at the recording site de-insulated with the exposed conductor. Depending on the size of the electrodes and cells, the recording of electrical activity may be from a single cell or from a cell population containing cell aggregates. Each electrode site is generally connected to the input side of a high input impedance, low noise amplifier with or without an AC coupling capacitor to amplify a relatively small extracellular signal. it can. Examples of such biosensors are Novak et al., IEEE Transactions on Biomedical Engineering BME-33 (2) (1986), 196-202; Drodge et al., J. Neuroscience Methods 6 (1986), 1583-1592; Eggers et al., Vac. Sci. Technol. B8 (6) (1990), 1392-1398; Martinoia et al., J. Neuroscience Methods 48 (1993), 115-121; Maeda et al., J. Neuroscience 15 (1995), 6834-6845; and Mohr et al., Sensors and Actuators B-Chemical 34 (1996), 265-269.

  Devices made and adapted to analyze the arrays described above are also the subject of the present invention.

  The cells, cell aggregates, tissues, organs and methods of the present invention are particularly suitable for use in drug screening and therapeutic applications. For example, the differentiated stem cells of the present invention can be used to screen for factors that affect the characteristics of differentiated cells (eg, solvents, small molecules, drugs, peptides, polynucleotides, etc.) or environmental conditions (eg, culture conditions or manipulations). it can. A specific screening application of the present invention relates to the testing of pharmaceutical compounds in drug research. It is generally referred to in the standard text “In vitro Methods in Pharmaceutical Research”, Academic Press, 1997 and US Pat. No. 5,030,015. Evaluation of the activity of a candidate pharmaceutical compound generally involves combining a differentiated cell of the invention with a candidate compound and the compound (when compared to an untreated cell or a cell treated with an inactive compound). Determining any change in cell morphology, marker phenotype or metabolic activity that is likely to be attributed, and then correlating the effect of the compound with the observed change. This screening can be done, for example, if the compound is designed to have a pharmacological effect on a particular cell type, or designed to have an effect elsewhere. This is the case when the compound will have unintended side effects. Two or more drugs can be tested in combination (by blending with the cells simultaneously or sequentially) to detect potential drug-drug interaction effects. In some applications, compounds are first screened for potential toxicity (Castell et al., Pp. 375-410 in “In vitro Methods in Pharmaceutical Research,” Academic Press, 1997). Cytotoxicity can be determined primarily by the effect on cell viability, survival, morphology, and expression or release of specific markers, receptors or enzymes. The effect of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [H] thymidine or BrdU incorporation, particularly at unscheduled points in the cell cycle, or at levels exceeding that required for cell replication, is consistent with drug effects. Further undesirable effects can include an abnormal rate of sister chromatid exchange as determined by metaphase elongation. Further details are mentioned in A. Vickers (pp 375-410 in “In vitro Methods in Pharmaceutical Research,” Academic Press, 1997).

Thus, in a further embodiment, the present invention provides:
(A) contacting a test sample comprising cells, cell aggregates, tissues or organs prepared according to the method of the present invention or differentiating with a test substance;
(B) determining a phenotypic response in the test sample when compared to a control sample, the change in phenotypic response in the test sample when compared to a control sample is Obtaining and / or profiling a test substance capable of influencing cell generation and / or tissue structure formation, comprising a step, which is an indication that the test substance has an effect on cell development and / or tissue structure formation On how to do.

  These methods can replace a variety of animal models and can form new human-based testing and extreme environment biosensors. Specifically, the methods of the invention can be used for in vitro testing for toxicity, mutagenicity and / or teratogenicity. Since the cells and tissues obtained according to the present invention are closer to the situation in vivo, the results obtained by the toxicity assay of the present invention are also predicted to correlate with the in vivo teratogenicity of the test compound. Is done.

  For example, compounds, particularly intracardiac active compounds, can be obtained from DE 195 25 285 A1; Seiler et al., ALTEX 19 Suppl. 1 (2002), 55-63; Takahashi et al., Circulation 107 (2003), 1912-1916, and Schmidt et al., Int. J. Dev. Biol. 45 (2001), 421-429, this latter reference describes ES cell heart and myogenic cells. Describes the ES cell test (EST) used in the European Union validation study for screening for embryotoxic substances by determining their differentiation into a concentration-dependent manner.

  Cells and tissues of the central nervous system (CNS) produced by the method of the present invention or during differentiation by the method are examined, for example, by cell culture such as that described in US Pat. No. 6,498,018. be able to. Similarly, cells and tissues related to the liver can be examined. See for example US application US2003 / 0003573. Based on post-differentiation multipotent embryonic stem (ES) cells derived from mice and rats using embryonic germ (EG) cells derived from primordial germ cells, detecting chemical inductive effects on embryonic development A further in vitro test method for examining differentiation for the purpose of embryotoxicity / teratogenicity screening is described in international application WO 97/01644 and can be adapted according to the teachings of the present invention.

  Suitable compound formulations for testing do not include additional ingredients such as preservatives that have a significant effect on the overall formulation. Thus, a suitable formulation consists essentially of a biologically active compound and a physiologically acceptable carrier such as water, ethanol, DMSO and the like. However, if a compound is a liquid without a pharmaceutical additive, the formulation may consist essentially of the compound.

  Furthermore, a differential response to various concentrations may be obtained by performing a plurality of assay methods in parallel at various compound concentrations. As is known in the art, determining the effective concentration of a compound typically uses a range of concentrations resulting from a dilution of 1:10 or other log scale. The concentration may be further subdivided by a second series of dilutions as needed. Typically, one of these concentrations is used as a negative control, ie zero concentration or below detection level.

  The compounds of interest encompass many chemical classes, but typically these are organic molecules. Candidate substances contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonds, and typically contain at least amine, carbonyl, hydroxyl or carboxyl groups, preferably of the functional chemical groups. Of at least two. Candidate substances often contain cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate substances are also found in biomolecules including peptides, nucleic acids, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

  Compounds and candidate substances are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Numerous means are available for random and directed synthesis of a wide range of organic compounds and biomolecules, including, for example, expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or are readily made. For example, inhibition of tumor-induced angiogenesis and matrix-metalloproteinase expression in face-to-face cultures of embryoid bodies and globular tumors by plant components used in classical Chinese medicine is Wartenberg et al., Lab. Invest. 83 ( 2003), 87-98.

  In addition, natural or synthetically generated libraries and compounds can be readily modified by conventional chemical, physical and biochemical means and used to create combinatorial libraries. Known analogs of pharmacological agents may be subjected to designated or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs.

  Further, the compound may be contained in a sample containing a fluid to which additional components such as components that affect ionic strength, pH, total protein concentration and the like have been added in advance. In addition, the sample may be processed to allow at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, such as under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow measurable detection, and usually about 0.1 μl to 1 ml of biological sample is sufficient.

  Test compounds include all classes of molecules described above, and may also include samples of unknown content. Many samples will contain a solution of the compound, but solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, such as groundwater, seawater, mine waste, etc .; lysates prepared from biological samples, such as grains, tissue samples, etc .; time courses during preparation of manufactured samples, such as pharmaceuticals; and for analysis A library of compounds to be prepared, etc. are included. Samples of the subject compound are evaluated for potential therapeutic value, ie drug candidates.

The test compound may optionally be a combinatorial library for screening a plurality of compounds. Such a collection of test substances can have a diversity of about 10 ^{3 to} about 10 ⁵ species, usually reduced sequentially in carrying out the method, optionally two or more times with others, Combined. Compounds identified by the methods of the present invention can be obtained in solution or after binding to a solid support by PCR, oligomer restriction (Saiki et al., Bio / Technology 3 (1985), 1008-1012 ), Allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sci. USA 80 (1983), 278), oligonucleotide ligation assay (OLA) (Landegren et al., Further evaluation, detection, cloning, sequencing and the like can be performed by any means commonly used for detection of specific DNA sequences such as Science 241 (1988), 1077). Molecular techniques for DNA analysis have been reviewed (Landegren et al., Science 242 (1988), 229-237). Thus, the method of the present invention can also be used for transcriptional profiling of embryonic and adult stem cells. See, for example, Ramalho-Santos et al., Science 298 (2002), 597-600; Tanaka et al., Genome Res. 12 (2002), 1921-1928.

Incubation includes conditions that allow contact between the test compound and ES cells or ES-derived cells. Contacting can be carried out both in vitro and in vivo conditions. For example, it may be preferable to examine an array of compounds or small molecules on one or few ES cells on a “chip” or other solid support. See above. For example, cardiomyocytes or neurons on the chip may be responsive to certain compounds and at a rate of contraction or firing, respectively, for detection of harmful or at least biologically active environmental agents. Will yield a number reading.

The neurological biocompatible electrode array allows stem cells to undergo further differentiation on the array itself. These arrays can measure real-time changes in electrical activity in ES cell-derived neurons in response to the presence of known or unknown agents. The electrical activity of cardiomyocytes can be observed by plating the cells on an array of extracellular microelectrodes (Connolly et al., Biosens. Biores. 5 (1990), 223-234). The cells show regular contraction and the recorded extracellular signal shows a relationship to intracellular voltage recording (Connolly et al., Supra). This non-invasive method allows long-term observation and is simpler and more robust than typical whole cell patch clamp techniques.

  Thus, in one preferred method of the invention, the phenotypic response to be determined preferably comprises an electrophysiological characteristic that is determined during the ongoing differentiation process. This embodiment is particularly suitable for providing modulation reference patterns and a database of modulation reference patterns for a wide range of biologically active compounds. The reference pattern is then used for test compound identification and classification. Evaluation of test compounds may be used to achieve a variety of results.

  Methods for classifying biological agents according to the spectral density signature of the changes induced by the intracellular potential are known to those skilled in the art. See, for example, US Pat. No. 6,377,057. Thus, biologically active compounds are classified according to their effect on ion channels, changes in membrane potential and ion current, and the frequency components of action potentials elicited by the compound in excitable cells. Such induced membrane potential or action potential spectral density changes are characteristic of each channel type modulated by the test compound. The pattern of spectral changes in membrane potential is determined by contacting responsive cells with the compound and observing membrane potential or ionic current over time. These changes correlate with the effect of the compound or class of compounds on the ion channels of responsive cells. This pattern of spectral change provides a signature unique to the compound and provides a useful method for characterization of channel modulators. The effect of a compound on ion channels and the action potential of living cells can provide useful information regarding the classification and type of the compound. Methods and means for extracting such information are particularly important for the analysis of biologically active compounds and have specific uses in pharmaceutical screening, drug discovery, environmental observation, biological war detection and classification, and the like. Examples of whole cell-based biosensors are Gross et al., Biosensors and Bioelectronics 10 (1995), 553-567; Hickman et al. Abstracts of Papers American Chemical Society 207 (1994), BTEC 76; and Israel et al., It is described in American Journal of Physiology: Heart and Circulatory Physiology 27 (1990), H1906-H1917.

  Connolly et al., Biosens. Biores. 5 (1990), 223-234 describes a planar array of microelectrodes developed to observe the electrical activity of cultured cells. This device can incorporate surface topographic features into the insulating layer above the electrodes. Semiconductor technology is used for the production of gold electrodes and the deposition and patterning of insulating layers made of silicon nitride. These electrodes were examined using a heart cell culture of chicken embryonic muscle cells, and the physical pulsation of the cultured cells was correlated with the obtained simultaneous extracellular voltage measurements.

  Molecular control of cardiac ion channels is described in Clapham, Heart Vessels Suppl. 12 (1997), 168-169. Oberg and Samuelsson, J. Electrocardiol. 14 (1981), 13942 performed a Fourier analysis on the repolarization phase of the cardiac action potential. Rasmussen et al., American Journal of Physiology 259 (1990), H370-H389, describes a mathematical model of electrophysiological activity in the atrium of an American edible moth.

  There is a great deal of literature on the entire ion channel field. A review of this document can be found in a series of books, Academic Press, "The Ion Channel Factsbook" by Edward C. Conley and William J. Brammar, 1-4th Edition. Overview of extracellular ligand-gate channel (ISBN: 0121844501), intracellular ligand-gate channel (ISBN: 012184451X), inward rectifier and intercellular channel (ISBN: 0121844528), and voltage-gate channel ( ISBN: 0121844536) to Hille, B. (1992) "Ionic Channels of Excitable Membranes", 2.sup.nd Ed. Sunderland MA: Sinauer Associates.

  In another aspect, cells cultured or modified using the materials and methods provided by the present invention are mounted on a support surface and screened for bioactive agents. In one example, the cells are connected to a substrate so that electrophysiological changes in the cells in response to external stimuli can be measured, eg, for use as a high throughput screen for biological agents. In addition, the cells can be transfected with DNA that targets, expresses or knocks out a specific gene or gene product within the cell. By providing a cell with such a chip attached to a measuring device such as a computer, a large number of compounds can be screened quickly and accurately. Furthermore, even if the cells or chips are connected to a measuring device arranged in an array, large-scale parallel screening will be possible.

  The assay of the present invention may be in a conventional laboratory format or may be adapted for high throughput. The term “high throughput” (HTS) refers to an assay design that can easily analyze multiple samples simultaneously and has the potential for robotic manipulation. Another preferred feature of the high throughput assay is an assay design that is optimized to reduce the amount of reagents used to achieve the desired analysis or to reduce the number of manipulations. Examples of assay formats include 96-well, 384-well or more well plates, floating droplets, and “love-on-a-chip” microchannel chips used for liquid handling experiments. Those skilled in the art will appreciate that as plastic molds and liquid scanning devices are miniaturized or better assay devices are designed, a larger number of samples will be performed using the design of the present invention. If you are familiar with it.

  In the method of the present invention, the cells are preferably contained in a container, such as in a well of a microtiter plate which may be a 24-, 96-, 384- or 1586-well plate. Alternatively, the cells can be introduced into a microfluidic device, such as that provided by Caliper (Newton, Mass., USA). In another preferred embodiment, the method of the present invention includes collecting 2, 3, 4, 5, 7, 10 or more measurements, optionally at different locations within the container. In certain embodiments of the screening methods of the invention, compounds known to activate or inhibit differentiation processes and / or tissue structure formation, such as retinoic acid, are added to the sample or medium. Again see above for suitable compounds.

  Furthermore, of course, the above-described methods can be combined with one or more steps of any one of the above-described screening methods, or other screening methods known in the art. Methods for discovery of clinical compounds include, for example, ultra-high throughput screening for lead identification (Sundberg, Curr. Opin. Biotechnol. 11 (2000), 47-53) and structure-based drug design (Verlinde and Hol , Structure 2 (1994), 577-587) and combinatorial chemical methods for lead optimization (Salemme et al., Structure 15 (1997), 319-324). Once a drug is selected, the method repeats the method used to make a rational drug design with the modified drug to determine whether the modified drug exhibits better affinity. There may be additional steps to evaluate according to interaction / energy analysis, etc. The method of the present invention may be repeated one or more times so that differences in the collection of compounds are successively reduced.

  Substances are metabolized after their in vivo administration to be removed either by excretion or by being metabolized to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459). Thus, instead of using the actual compound or drug identified and obtained according to the method of the present invention, a prodrug of the corresponding formulation that is converted to its active form by his / her metabolism within the patient Can be used as Precautionary measures that may be paid for the use of prodrugs and drugs are described in the literature. For reviews, see for example Ozama, J. Toxicol. Sci. 21 (1996), 323-329.

  Furthermore, the present invention relates to isolated and / or produced compounds identified by any one of these methods for the treatment of abnormalities related to damaged or abnormal tissue or organogenesis, heart failure, etc. It relates to the use in the preparation of a therapeutic composition. See also above. Preferably, the isolated compound or the corresponding drug assists wound healing and / or healing of damaged tissue. As a method of treatment, the identified substance or a composition containing the same can be administered to a subject suffering from such an abnormality. The isolated and / or produced compounds identified by the methods described above can also be used as lead compounds for drug discovery and preparation of drugs or prodrugs. This usually includes the step of modifying the lead compound or derivative thereof, or an isolated compound as described herein, for example biologically modifying the substance to cause toxicity. Including altering, removing and / or derivatizing a portion thereof that is expected to increase availability, solubility and / or half-life. The method may further comprise mixing the isolated or modified material with a pharmaceutically acceptable carrier. The various steps cited above are known in the art. For example, computer programs for implementing these techniques can be used, such as Rein, Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York, 1989). Methods for preparing chemical derivatives and analogs are known to those skilled in the art, such as Beilstein, Handbook of Organic Chemistry, Springer Edition New York Inc., 175 Fifth Avenue, New York, NY 10010 USA, and Organic Synthesis, Wiley, New Explained in York, USA. Furthermore, suitable derivative and analog peptide mimetics and / or computer aided designs can be used, for example, according to the methods described above. Methods for lead generation in drug discovery further include the use of proteins, mass spectrometry (Cheng et al., J. Am. Chem. Soc. 117 (1995), 8859-8860) and some nuclei. Detection methods such as magnetic resonance (NMR) methods (Fejzo et al., Chem. Biol. 6 (1999), 755-769; Lin et al., J. Org. Chem. 62 (1997), 8930-8931) included. These are further analyzed by quantitative structure-action relationship (QSAR) analysis (Kubinyi, J. Med. Chem. 41 (1993), 2553-2564, Kubinyi, Pharm. Unserer Zeit 23 (1994), 281-290), combinatorial Biochemistry, classical chemical methods and others (see, for example, Holzgrabe and Bechtold, Pharm. Acta Helv. 74 (2000), 149-155) may be included or relied upon. In addition, examples of carriers and methods of formulation may be found at Remington's Pharmaceutical Sciences.

  Once a drug is selected according to any one of the above-described methods of the present invention, the drug or its prodrug can be synthesized in a therapeutically effective amount. The term “therapeutically effective amount” as used herein refers to a meaningful benefit for the patient, ie treatment, healing, prevention or amelioration of damaged tissue or treatment, healing, prevention or amelioration of such a condition. It means the total amount of the drug or prodrug sufficient to show an increase in speed. In addition or alternatively, particularly with respect to preclinical testing of drugs, the term “therapeutically effective amount” includes the total amount of drug or prodrug sufficient to elicit a physiological response in a non-human animal test. It is.

  The present invention further comprises a vector or vector composition as hereinbefore described, a pluripotent or multipotent cell, optionally a medium, a recombinant nucleic acid molecule, a standard compound and the like. It also relates to kit compositions containing specific reagents useful for performing any one of the methods described above, such as those previously described herein. Such a kit will typically include a compartmented carrier suitable for holding at least one container firmly. The carrier will further contain reagents useful for performing the method. The carrier may contain detection means such as a labeled enzyme substrate.

  Therefore, the means and methods of the present invention described hereinabove can be used in a variety of applications, including but not limited to “functions” using ES cells containing homozygous mutations of specific genes. Loss "assay," hyperfunction "assay using ES cells overexpressing exogenous genes, in vitro teratogenic / embryotoxic compound development analysis, pathologic cell function pharmacological assay and model system And the application of differentiation factors and growth factors for the induction of selectively differentiated cells that can be used as a source of tissue grafts. For reviews, see for example Guan et al., Altex 16 (1999), 135-141.

  These and other embodiments are disclosed and are included in the description and examples of the present invention. Further literature on any one of the substances, methods, uses and compounds used in accordance with the present invention could be retrieved from public libraries and databases, for example using electronic devices. For example, the National Center for Biotechnology Information of the National Institutes of Health and / or the public database “Medline” hosted by the National Library of Medicine may be used. Additional databases and web addresses are known to those skilled in the art, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL), and using Internet search engines Can also be obtained. An overview of patent information in biotechnology and a search for relevant sources of patent information useful for retrospective searching and current knowledge can be found in Berks, TIBTECH 12 (1994), 352-364.

  The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples and drawings, which are provided for the purpose of illustration only and are intended to be illustrative of the present invention. It is not intended to limit the scope of It is expressly stated that the contents of all cited references (including documents cited throughout this application, issued patents, published patent applications, manufacturer specifications, instructions, etc.) are incorporated herein by reference. However, none of the cited documents is admitted to be a de facto prior art to the present invention.

  In carrying out the present invention, unless otherwise specified, conventional techniques of cell biology, cell culture, molecular biology, gene transfer biology, microbiology, recombinant DNA, and immunology, which are levels of skill in the art, are used. Will use it.

  For further details of the general technique on stem cell technology, the practitioner should review standard texts and reviews such as Teratocarcinomas and embryonic stem cells: A practical approach (EJ Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (PM Wasserman et al., eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (Wiles, Meth. Enzymol. 225 (1993), 900,); Properties and uses of Embryonic Stem Cells: Reference can be made to Prospects for Application to Human Biology and Gene Therapy (Rathjen et al., Reprod. Fertil. Dev. 10 (1998), 31,). Stem cell differentiation has been reviewed by Robertson, Meth. Cell Biol. 75 (1997), 173; and Pedersen, Reprod. Fertil. Dev. 10 (1998), 31. In addition to the stem cell sources already mentioned above, further references are provided. Evans and Kaufman, Nature 292 (1981), 154-156; Handyside et al., Roux's Arch. Dev. Biol., 196 (1987), 185-190; Flechon et al., J. Reprod. Fertil. Abstract Series 6 (1990), 25; Doetschman et al., Dev. Biol. 127 (1988), 224-227; Evans et al., Theriogenology 33 (1990), 125-128; Notarianni et al., J. Reprod. Fertil. Suppl., 43 (1991), 255-260; Giles et al., Biol. Reprod. 44 (Suppl. 1) (1991), 57; Strelchenko et al., Theriogenology 35 (1991), 274; Sukoyan et al. , Mol. Reprod. Dev. 93 (1992), 418-431; Iannaccone et al., Dev. Biol. 163 (1994), 288-292.

  Molecular genetics and genetic engineering methods are generally described in the current version of Molecular Cloning: A Laboratory Manual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory. Press); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (FM Ausubel et al., eds.); and Recombinant DNA Methodology (R. Wu ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, vols. 154 and 155 (Wu et al. Eds.); Immobilized Cells And Enzymes (IRL Press, 1986 ); B. Perbal, A Practical Guide To Molecular Cloning (1984); Paper, Methods In Enzymology (Academic Press, Inc., NY); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell eds., 1986). Reagents, cloning vectors and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech. General techniques in cell culture and media collection include Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73 Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251). Other observations about the media and their effects on the culture environment have been made by Marshall McLuhan and Fred Allen.

Examples Example 1: Generation of transgenic ES cell clones for drug selection of ES cell-derived cardiomyocytes
Vector design Heart specific α-myosin heavy chain (αMHC) (GenBank accession number: U71441; Subramaniam et al., J. Biol. Chem. 266 (1991), 24613-24620; Sanbe et al., Circ. Res. 92 (2003), 609-616), the 5.5 kb BamHI-SalI fragment of the promoter region, the coding region of the puromycin resistance cassette (Pac), and the human cytomegalovirus (CMV) early promoter (P _CMV IE) After cutting with AseI-Eco47 III, it was inserted into the multicloning (MCS) site of the pIRES2-EGFP vector (Clontech ^(R) ). Within the resulting bicistronic vector (pαPIG), the heart-specific αMHC promoter drives the expression of both Pac as a drug selection marker and enhanced green fluorescent protein (EGFP) as a live reporter gene. The IRES (internal ribosome entry site) sequence provides separate translation of both proteins in stably transfected cells. The vector further contains kanamycin- and neomycin resistance cassettes for selection of transfectants in bacterial and ES cell cultures, respectively.

Transfection and selection of ES cell clones
5 × 10 ⁶ ES cells (strain D3: Doetschman et al., J. Embryol. Exp. Morph. 87 (1985), 27-45) were charged with 30 μg of DNA of pαPIG vector linearized with SacI. It was injected by the perforation method. Cells were seeded on 10 cm tissue culture dishes containing monolayers of neomycin resistant feeder cells inactivated with mitomycin. Forty-eight hours after seeding, neomycin (G418) 300 μg / ml was added to the medium to select stably transfected ES cell clones. Eight to ten days after the start of selection, colonies of viable ES cells were picked, trypsinized, and grown in order on 48-well, 24-well and 6 cm plates. The resulting clone was used in a cardiac differentiation protocol for screening.

  Differentiation was performed according to a standard “hanging drop” protocol as described, for example, in Maltsev et al., Circ. Res. 75 (1994), 233-244. From day 8 to day 10 of development, beating embryoid bodies (EB) expressing EGFP fluorescence were treated with 10 μg / ml puromycin. Puromycin cell death is already evident at 12 hours after treatment, at which point, among the number of clones, a pulsating agglomeration of EGFP positive cells not only survived the treatment but also a strong beat. The activity rate was shown. Already 3 to 5 days after treatment, EGFP positive cell clumps that were beating strongly accounted for the major proportion of cells in plated EBs and suspension cultures of EBs.

  Two clones (αPIG10 and αPIG44) that showed heart-specific expression of both EGFP and puromycin resistance cassettes were selected and used in further experiments.

Example 2: Co-culture of purified ES cell-derived heart cells and mouse embryonic fibroblasts Seven to ten days after puromycin treatment, beating EGFP-positive clumps of heart cells were collected by centrifugation and washed with PBS. Washed twice and treated with 0.1% collagenase B (Boehringer Mannheim) for 20 minutes at 37 ° C. After 10 minutes and at the end of the incubation, the cell suspension was gently pipetted through a blue tip of a 1 ml pipette. As a result, 1, 2 and 2 volumes again containing 20% fetal calf serum (FCS) are added, the cells are centrifuged, washed twice with this medium, resuspended, and fluorescent microscope Calculated below.

Mouse embryonic fibroblasts were obtained from 14-16 day old embryos according to standard procedures. See, for example, Joyner AL Gene targeting. A Practical Approach. Oxford University Press, 1993. Cells were grown to confluence, treated with 0.05% trypsin, washed twice with medium containing 20% FCS and calculated. For co-culture, approximately 50 × 10 ³ to 100 × 10 ³ fibroblasts are mixed with an equal amount of purified EGFP-positive ES cell-derived cardiomyocytes and placed on one well of a × 24 well plate. Plated or, depending on the experiment, plated on a multi-electron array (MEA). As shown in FIG. 4, one day after co-seeding, the fibroblasts formed a monolayer, but the EGFP-positive cardiomyocytes showed only one or a group of cells slightly attached to the fibroblasts. During the following 1 to 2 days, ES cell-derived EGFP positive cardiomyocytes showed complete integration and alignment with fibroblasts and acquired vertical morphology and orientation relative to surrounding fibroblasts (FIG. 4). . Cardiac cells integrated into embryonic fibroblasts showed viability and contractility for at least 1 or 2 weeks, as shown in MEA experiments (FIG. 5). For extracellular recording assisted by multi-electrode arrays (MEA), ES cell-derived cardiomyocytes and fibroblasts are glass with 60 titanium nitride electrodes (diameter 30 μm, spacing 200 μm) in the center of the MEA The cells were cultured on a multi-electrode array (MEA; Reutlingen, Germany, Multi Channel Systems) consisting of a substrate (5 cm × 5 cm) and an inner reference electrode. Extracellular electrophysiological recordings from cardiomyocytes were performed with the MEA60 system (Reutlingen, Germany, Multi Channel Systems). This system is for recording measurement data with MEA-1060 amplifier (bandwidth 10 Hz to 3 kHz; amplification: 1200), temperature controller HC-X to maintain the medium at 37 ° C, and MC rack software. Including computer systems. The recording sample rate was 4 kHz.

Example 3: Co-transplantation of purified ES cell-derived heart cells and mouse embryonic fibroblasts In order for mouse strain SV129 to be compatible with the origin of ES cell clones used to generate cardiomyocytes, standard techniques (eg, Joyner AL Gene targeting. A Practical Approach. See Oxford University Press, 1993). 50 × 10 ³ to 100 × 10 ³ purified cardiomyocytes and fibroblasts were mixed, and the cold infarcted heart of SV129 mice was mixed with Roell et al., Circulation 105 (2002), 2435-2441. Injections were made as described. Cardiomyocytes exhibiting both EGFP fluorescence and striated were detected in the transplanted heart within the time frame 10 to 70 days after the operation (FIG. 6), so that the survival of the transplanted ES cell-derived heart cells Power was supported.

Example 4: Basic design of transgenic ES cell clones for tissue modeling
Vector design:
The basic elements of a vector are cell type-specific genomic regulatory sequences (so-called “promoters”) that contain a common promoter and specific enhancer sequences. Typically they also contain intron-exon fragments that extend upstream of the gene coding region and sometimes are not translated. The promoter determines the cell specific activation of the drug resistance cassette, which is the second basic element of the vector and usually follows from this latter downstream to the right of the promoter. This combination allows removal of undifferentiated ES cells along with differentiating cells towards a cell type unrelated to the target cell type that activates the drug resistance cassette during the differentiation process. it can.

  In addition, it is recommended to include in the vector a so-called live color fluorescent protein cassette joined to the drug resistance cassette via an internal ribosome entry site (IRES). Such a bicistronic vector makes it possible to transcribe both a drug resistance gene cassette and a live reporter gene cassette from the same vector under one cell type-specific promoter. IRES then allows for independent ribosome translation of both cassettes and visualizes certain differentiated cells for observation. To date, at least three different forms of enhanced green fluorescent protein (EGFP)-EYFP (yellow), ECFP (blue) and hcRFP (red)-are visible simultaneously in at least three different cell types in the same culture. Can be used to The basic design of such a vector is shown in FIG.

Transgenic ES clone:
The core of the method of the present invention is the drug selection of the cell types constituting the target tissue in parallel in one cultured strain of ES cells being differentiated. The advantage of such an approach is that the interaction between purified cell types is released from unrelated cells immediately after using the natural cue to “crosstalk” signaling. This is the point that a viable tissue-like structure is formed. Two variations of such an approach are provided:
a) Multiple transgenic ES cell clones stably transfected with a specific number of vectors having a drug selection cassette operated by a specific promoter depending on the cell type constituting the desired tissue type. In such a variant, all the cell types that appear originate from a common progenitor ES cell clone, and the resulting ratio between the different cellular components depends on the respective relative differentiation rate of each (Fig. 2A and 3B);
b) Chimeric embryoid body (EB): This approach results in a number of transgenic ES cell clones, each one having a drug resistance cassette that is driven by one of the cell type specific promoters. Has only one vector. For tissue modeling, the relevant clones must be mixed at an early stage of differentiation (“hanging drop” or “mass culture”) to form ES cell aggregates (EBs), in which case After drug selection, the appearing cell types originate from different corresponding ES cell clones, and the final ratio between the cellular components is still dependent on and controlled by the initial ratio between the different ES cell lines. Yes (FIGS. 2B and 3C).

Example 5: Cardiac tissue modeling in ES cell lines A system for drug selection of ES cell-derived cardiomyocytes based on the basic scheme of the bicistronic vector described above was established. For this purpose, the α-myosin heavy chain heart-specific promoter (αMHC promoter) and the puromycin resistance cassette are referred to as “cell type-specific promoter” and “drug resistance cassette for cell type selection”, respectively (see FIG. 1 and 3A) were inserted into the vector pIRES2-EGFP (Clontech ^(R) ) having IRES and enhanced green fluorescent protein (EGFP) as “IRES” and “living fluorescent reporter cassette” (FIG. 1), respectively. . See also Examples 1 and 2 of international application WO02 / 051987. This system allows for rapid and efficient purification of viable cardiomyocytes suitable for transplantation. The obvious advantages of this system are demonstrated by the ability to observe differentiation, heart-specific selection and the fate of transplanted cells. Furthermore, it was also shown that puromycin-purified cardiomyocytes fully integrated and aligned with embryonic fibroblasts within one or two days of co-culture (FIG. 4). In such co-cultures, ES cell-derived purified cardiomyocytes remain in good functional state for at least two weeks, during which both spontaneous contraction and external potential (FA) signals are high. Recorded through electrode array (MEA) measurements (Figure 5).

  Fibroblasts are known to be key cellular elements of connective tissue in mammalian and non-mammalian species. Especially in the mouse heart, they constitute up to 50% of the embryonic heart and up to 80% in the adult heart. Another important non-cardiac element of heart tissue is provided by endothelial cells as the primary cellular element for capillaries and blood vessels with important nutritional functions. Thus, it is expected that ES cell-derived hearts, endothelium and fibroblasts can constitute a complete set to form heart-like tissue.

Vector and ES cell clone design:
1) In the case of heart-specific vectors, the above αMHC promoter can be used, or other heart-specific promoters (MLC2v, MLC1a, MLC2a, β-MHC, etc.) can be used as “cell type-specific promoters” and enhanced cyanide Fluorescent protein (ECFP, Clontech®) can be used as a live reporter gene, along with IRES and puromycin (or some other selectable marker) cassette, according to FIG. 3A.

2) In the case of endothelial cell-specific vectors, Tie2 (or other endothelial cell-specific promoters such as Tie1, cadherin, etc.) is referred to as a “cell type-specific promoter” and enhanced yellow fluorescent protein (EYFP, Clontech ^(R) ) can be used as a live reporter gene, along with IRES and puromycin (or some other selectable marker) cassette, according to FIG. 3A.

3) In the case of fibroblast-specific vectors, collagen I (or other fibroblast-specific promoter) was designated as the “cell type-specific promoter” and hcRed fluorescent protein (hcRFP, Clontech ^(R) ) was alive As a reporter gene, it can be used according to FIG. 1 together with an IRES and puromycin (or some other selectable marker) cassette.

  ES cell clone design, differentiation and selection schemes can be performed according to the two main principles described above: with three vectors—one clone ”(FIG. 3B) or three vectors—three clones (FIG. 3C). is there. Transfection and selection of ES cell-derived cell types and transplantation are performed as described in International Application WO 02/051987, the disclosure of which is incorporated herein by reference. Specifically, see Examples 1 and 2 of WO02 / 051987 and the references cited therein.

  By using three different live fluorescent reporter vectors, differentiation, selection and cell-to-cell connections during tissue formation in a single culture can be tracked in a “live” mode. In vitro formation of heart tissue-like structures in ES cell lines can be used as an important physiological system for the examination of various cardiotrophic and cardiotoxic agents in biochemical and electrophysiological (MEA) experiments. In addition, it may be an important source of transplant material in heart disease replacement therapy.

Example 6 Dual Transgenic System for Cardio-Vascular Selection in ES Cell Lines The main purpose of this experiment was to have two ES cells closely related to each other both in terms of function and common mesodermal origin It was a parallel selection of derived cell types. For this purpose, stable double transgenic ES cell clones were generated. In this clone, one vector has a drug resistance cassette under the control of a cardiac specific promoter, while the second has both a drug resistance and a live fluorescent reporter cassette under the control of an endothelial cell specific promoter. To have. Heart and endothelial cells appear very early in actual embryogenesis and constitute a functional and anatomically related element for the developing heart. Thus, because they are effectively selected from a single culture of differentiating ES cells, these cell types show a pattern of self-assembly that operates by cues similar to those occurring during actual embryonic heart development Predicted to be. In the first experimental example, endothelial cell-like cells were identified with enhanced green fluorescent protein (EGFP) fluorescence and colorless cardiomyocyte-like cells had to be identified by detection of contractile conglomerates. In further experiments, chimeric embryoid bodies were made that consisted of both the clones described above and another transgenic clone with a red fluorescent protein (HcRFP) both driven by a heart-specific promoter and a drug resistance cassette. Thus, the latter experiment was able to visualize the differentiation and selection of both cardiac and endothelial cell types.

Vector:
1) For vector pαMHC-pac, puromycin resistance cassette (Pac) was excised from pCre-Pac vector (Taniguchi et al., Nucleic Acid Research 26 (1998, 679-680) with Hind III-Sal I restriction enzyme, The EGFP cassette was deleted from the αMHC-EGFP vector with the BamH I-Afl II enzyme and then blunt-ended ligated, and a vector obtained by linearization with Hind III was used for injection by electroporation of ES cells.
2) For the vector pTie2 -Pac-IRES-EGFP (pTie2-PIG), the Pac-IRES-EGFP cassette was excised from the pPIG vector with Sal I-Afl II and the pSPTg.T2FXK vector (Schlaeger et al., Proc. Natl. Acad. Sci. USA 94 (1997), 3058-3063) was inserted into the NotI site between the Tie2 promoter and the Tie2 enhancer by blunt end ligation.

For injection of ES cells by electroporation, the Tie2 promoter-PIG-Tie2 enhancer fragment was excised from the resulting vector with Sal I and purified by electrophoresis on a 1% agarose gel.
3) For the vector pαMHC-hcRFP, a 5.5 kb heart αMHC promoter fragment was excised from pαMHC-BS2SK ⁺ (Robbins, Trends Cardiovasc. Med. 7 (1997), 185-191) with BamH I-SaI I, pHcRed1 -1 (Clontech ^(R) , USA ⁾ was blunt-ended at the SmaI site.

ES cell culture, transformation and differentiation protocols:
ES cells were cultured and electroporated as described (Kolossov et al., J. Cell Biol. 143 (1998), 2045-2056). 5 × 10 ⁶ ES cells (D3 strain) were co-transfected with 30 μg of DNA each of pαMHC-Pac and Tie2 promoter-PIG-Tie2 enhancer fragment. G418 resistant clones were selected, expanded and differentiated as described (Kolossov et al., J. Cell Biol. 143 (1998), 2045-2056). For the production of chimeric EBs, cell suspensions from transgenic clones Tie2-PIG / αMHC-Pac and αMHC-hcRFP / αMHC-Pac were used for each clone with a maximum of 0.01 × 10 ⁶ cells per ml. Mixed to density (200 cells of each clone per drop).

  EB was observed with a fluorescence microscope Axiovert 200M (Zeiss, Germany).

Clone Tie2-PIG / αMHC-Pac:
Spontaneous contraction began on days 8-10 of development. On days 11-14, the first EGFP positive cells were detected only in pulsatile EBs in areas that overlap or are very close to the contractile heart mass. At this point puromycin (5 μg / ml) was added, and then the medium was changed every 2-3 days. During the next day, increased contraction of the heart conglomerate was detected with increased EGFP expression. At the same time, intensive death of puromycin non-resistant cells was observed. Typically, already 4 days after puromycin treatment, EGFP positive cells formed a network embedded within actively beating conglomerates of heart cells. Fluorescence intensity increased dramatically 10 days after puromycin treatment and beyond.

Chimera EB: Clone Tie2-PIG / αMHC-Pac + Clone αMHC-hcRFP / αMHC-Pac:
Similar to the EBs from the clones described above, chimeric EBs showed EGFP expression over the same time course in the beating area. At the same time, strong RFP fluorescence was detected in the same beating area and became a marker for differentiated cardiomyocytes. Surprisingly, the EGFP and RFP fluorescent agglomerates spatially overlapped but did not completely overlap with the pulsatile agglomerates that exhibited a bright green-red mosaic structure. Both green and red fluorescence increased significantly during puromycin treatment.

  Thus, the experiments described above clearly demonstrate a clear and strong relationship between cardiac differentiation and endothelial cell differentiation in ES cell lines. EBs without contractile activity did not express any EGFP fluorescence. Most areas of EGFP-expressing cells were very close to pulsatile clustering, or showed high spatial agreement with both cell types, as they overlapped completely. The relationship between these two cell types became apparent after puromycin treatment. After the majority of undifferentiated cells died, the network of EGFP fluorescent cells was embedded within the beating heart mass, often showing signs of structural orientation. Surprisingly, the proliferation of endothelial cells after release from undifferentiated ES cells, as demonstrated in the present invention with respect to cardiac cells, was that the intensity of EGFP fluorescence increased dramatically during puromycin treatment. Was implied.

  Based on the close relationship between cardiac and endothelial cell elements, these structures were differentiated into differentiating multi-transgenic ES cell cultures, especially as evident in multicolor fluorescent images of beating clusters. It can be considered as a prototype of a cardiovascular tissue-like structure produced by means of selecting a drug from

  Finally, the presented data point to the basic feasibility of “tissue modeling” through multiple lineage selection in multiple transgenic ES cell lines.

  Those skilled in the art will recognize that the compositions and techniques provided in the description section can be effectively modified without departing from the spirit of the invention as embodied in the following claims. Like.

FIG. 1 shows the main scheme of a vector for tissue modeling in the ES cell line of the present invention. FIG. 2 shows two vectors: (A) one transgenic ES cell clone; (B) two transgenic ES cell clones. FIG. 3 shows three vectors: (A) vector construct; (B) one transgenic ES cell clone; (C) three transgenic ES cell clones. FIG. 4 shows that ES cell-derived puromycin-selected EGFP ⁺ cardiomyocytes were co-seeded with mouse embryonic fibroblasts. A, B-1, 5d; C and D-6d in co-cultures. The alignment of EGFP ⁺ cardiomyocytes with fibroblasts on days 5 and 6, respectively, is evident. FIG. 5 shows that mouse embryonic fibroblasts and EGFP-positive ES cell-derived cardiomyocytes purified with puromycin were separated by collagenase treatment and co-seeded on MEA. EGFP-positive heart clusters that beat on day 4 after seeding were fully integrated on the fibroblast layer (A) and regular FP was recorded from most of these (B). FIG. 6 shows that ES cell-derived cardiomyocytes selected with puromycin were successfully transplanted into the cold infarcted area when co-transplanted with syngeneic fibroblasts. A shows the heart 40 days after transplantation viewed under complex transmission fluorescence. In B and C, it can be seen that the transplanted EGFP positive (C) ES cell-derived cardiomyocytes show a striated pattern after alpha-actinin immunostaining (B).

Claims (81)

Culturing a first cell type derived from embryonic stem (ES) cells in the presence of at least one embryonic second cell type; integrating and aligning said at least two cell types into a tissue or tissue-like structure; A method of modeling and / or obtaining a tissue or tissue-like structure. The method of claim 1, wherein the ES cell of the first cell type derived from the ES cell comprises a selection marker operably linked to a first cell type specific regulatory sequence specific to the first cell type. . The method of claim 2, wherein the selectable marker provides resistance to puromycin. The ES cell of the first cell type derived from the ES cell comprises a reporter gene operably linked to a first cell type-specific regulatory sequence specific to the cell type. The method described in 1. 5. The method of claim 4, wherein the cell type specific regulatory sequence of the reporter gene is substantially the same as the first cell type specific regulatory sequence of the marker gene. 6. The method of claim 5, wherein the reporter is selected from various color forms of enhanced green fluorescent protein (EGFP). The method according to any one of claims 4 to 6, wherein the marker gene and the reporter gene are contained on the same recombinant nucleic acid molecule. The method according to claim 7, wherein the marker gene and the reporter gene are contained on the same cistron. The first cell types are neuronal cells, glial cells, cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes, chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts 9. The cell according to any one of claims 1 to 8, selected from the group consisting of cells, keratinocytes, dendritic cells, hair follicle cells, renal canal epithelial cells, vascular endothelial cells, testicular progenitor cells, smooth muscle and skeletal muscle cells. Method. The method according to claim 1, wherein the first cell type is a cardiomyocyte. 11. The method of claim 10, wherein the first cell type specific regulatory sequence is atrial and / or ventricular specific. The method according to claim 10 or 11, wherein the at least one embryonic second cell type is a fibroblast or an endothelial cell. 13. The method according to any of claims 1 to 12, further comprising culturing the at least two cell types in the presence of a third cell type derived from embryonic or embryonic stem (ES) cells. 14. The method of claim 13, wherein the third cell type is an endothelial cell or fibroblast. Co-cultured cells as defined in any of claims 1-14. A tissue obtained by the method according to any one of claims 1 to 14. (A) a cell inoculum comprising a co-cultured cell of claim 15 or a tissue of claim 16 wherein differentiation has been induced in at least a portion of a previously damaged tissue area. Introducing, steps and:
(B) transplanting the introduced cellular inoculum in situ as viable cells or tissues placed in the previously damaged tissue section, wherein the feeding results in the feeding Improving the tissue and / or organ function in the mammal, comprising improving the tissue and / or organ function of the animal. (A) An undifferentiated mammalian embryonic stem (ES) cell containing a resistance gene and a reporter gene under the control of the same heart-specific promoter is cultured in vitro in a medium containing a selective agent for the resistance gene. And culturing under conditions that allow differentiation of the ES cells into cardiomyocytes;
(B) isolating the differentiated cardiomyocytes and / or pre-differentiated cells, optionally together with cells that are differentiating from the cardiomyocytes toward an unrelated cell type during the differentiation process Removing the step;
(C) then co-implanting the cardiomyocytes with embryonic or ES cell derived fibroblasts into at least a portion of a previously infarcted area of heart tissue;
(D) transplanting the introduced cellular inoculum in situ as viable cells placed in a previously infarcted area in the heart tissue, wherein the transplantation results in the mammalian A method for improving cardiac function in a mammal after myocardial infarction, comprising the step of improving cardiac function. The method according to claim 18, wherein the resistance gene and the reporter gene are contained in a bicistronic vector and separated by IRES. 20. The method of claim 19, wherein the resistance gene provides resistance to puromycin, the marker is EGFP, and the promoter is a cardiac αMHC promoter. (A) transfecting one or more pluripotent or multipotent cells with a recombinant nucleic acid molecule comprising first and second cell type specific regulatory sequences operably linked to at least one selectable marker. The second cell type is different from the first cell type;
(B) culturing the cell under conditions allowing differentiation of the cell;
(C) isolating cells of at least two differentiated cell types and / or cells that have not differentiated, selectively independent of the cell type of interest that activates the selectable marker in the course of differentiation Removing together with the differentiating cells towards the cell type;
A method of modeling and / or obtaining a tissue or tissue-like structure comprising: Transfecting said one or more cells with a recombinant nucleic acid molecule comprising at least one additional cell type-specific regulatory sequence operably linked to at least one selectable marker comprising said at least one cell 24. The method of claim 21, further comprising the step of further cell types being different from the first and second cell types. 23. The method of claim 21 or 22, wherein the cell is an embryonic stem (ES) or embryonic germ (EG) cell. 24. A method according to any of claims 21 to 23, wherein the recombinant nucleic acid molecules are contained within the same vector or different vectors. The cell types are neuronal cells, glial cells, cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes, chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts, 25. A method according to any of claims 21 to 24, selected from the group consisting of keratinocytes, dendritic cells, hair follicle cells, kidney duct epithelial cells, vascular endothelial cells, testicular progenitor cells, smooth muscle and skeletal muscle cells. The method according to claim 7 or 8, wherein the promoter is selected from the group consisting of αMHC, MLC2V, caterin, Tie-2 and a collagen promoter. 27. A method according to any of claims 21 to 26, wherein the one or more recombinant nucleic acid molecules are transfected into the one or more cells simultaneously or sequentially. 27. At least two different cells or clones thereof are transfected and selected, wherein said at least two different cells or cell clones contain recombinant nucleic acid molecules having different cell type specific regulatory sequences. The method according to any one. 29. The method of claim 28, wherein the at least two different cells or cell clones are mixed at an early stage of differentiation to allow cell aggregate formation. 30. The method of claim 29, wherein the cell aggregate is a chimeric embryoid body (EB). 31. Any of the claims 21-30, wherein the cell or one of its cell clones is transfected and selected, wherein the cell or cell clone contains a recombinant nucleic acid molecule having at least two different cell type specific regulatory sequences. The method of crab. 32. A method according to any of claims 21 to 31, wherein at least two of the selectable markers operably linked to the different cell type specific regulatory sequences are identical. 33. Any of the selection markers operably linked to the different cell type specific regulatory sequences results in resistance to puromycin, bleomycin, hygromycin, methotrexate, or neomycin. The method described. 34. The method of any of claims 21-33, wherein one or more of the recombinant nucleic acid molecules further comprises a reporter operably linked to the cell type specific sequence. 35. The method of claim 34, wherein the reporter is selected from various color forms of enhanced green protein (EGFP). 36. The method of claim 35, wherein EYFP (yellow), ECFP (blue) and / or hcRFP (red) are operably linked to different cell type specific sequences. 37. A method according to any of claims 34 to 36, wherein the selection marker and the reporter are expressed from a bicistronic vector. 38. The method of claim 37, further comprising one or more internal ribosome entry sites (IRES), wherein the IRES separates the selectable marker and the reporter. 39. A method according to any of claims 21 to 38, further comprising the step of causing self-assembly of different cell types. 40. A method according to any of claims 1 to 14 or 21 to 39, further comprising the step of analyzing the physiological and / or developmental status of the cell or cell aggregate. 41. The method of claim 40, wherein the situation is analyzed by observing differentiation of the electrical activity of the cells on the array. 42. The method of claim 41, wherein the situation is analyzed by recording extracellular field potential with a microelectrode array (MEA). 43. One or more cells obtained by the method according to any of claims 21 to 42, wherein the one or more cells are capable of differentiating into at least two cell types. A cell aggregate of at least two different cell types obtained by the method according to any of claims 21 to 42. A tissue obtained by the method according to any one of claims 1 to 42, or comprising the cell according to claim 43 or the cell aggregate according to claim 44. 45. An organ comprising the cell according to claim 43, the cell aggregate according to claim 44, or the tissue according to claim 45. 45. An implant or graft comprising a cell according to claim 43, a cell aggregate according to claim 44, a tissue according to claim 45, or an organ according to claim 46. A composition of a substance comprising the recombinant nucleic acid molecule according to any one of claims 21 to 42, the cell according to claim 43, the cell aggregate according to claim 44, or the tissue according to claim 45. . 43. Use of a method according to any of claims 1 to 14 or 21 to 42 for analyzing the initial stages of tissue formation during embryonic development or the influence of factors and compounds on this process. 45. A subject in need of treatment of a damaged tissue or organ, the cell of claim 43, the cell aggregate of claim 44, the tissue of claim 45, or the organ of claim 46. A method of treating a damaged tissue or organ in a subject comprising the step of implanting or transplanting into the subject. (A) a mammalian embryonic stem cell (ES) cell that contains a resistance gene under the control of heart, fibroblast and / or endothelial cell specific regulatory sequences and is under said same specific regulatory sequence; Transfecting the recombinant nucleic acid molecule, optionally also including one or more reporters;
(B) The ES cells are cultured in vitro in a medium containing a selective substance for the resistance gene under conditions that allow the ES cells to differentiate into cardiomyocytes, fibroblasts and / or endothelial cells. A step to do;
(C) removing undifferentiated cells from the differentiated cardiomyocytes, fibroblasts and / or endothelial cells, optionally together with differentiating cells towards an irrelevant cell type;
(D) aligning and integrating the differentiating cardiomyocytes, fibroblasts and / or endothelial cells with heart-like tissue as an optional step;
(E) then co-implanting the cardiomyocytes, fibroblasts and / or endothelial cells or the tissue into at least a portion of a previously infarcted area of heart tissue;
(F) transplanting the introduced cells or tissues in situ as viable cells located in the previously infarcted area in heart tissue, the result of the transplant being the cardiac function of the mammal Improving the cardiac function of the mammal after myocardial infarction, comprising: improving. 52. The method of claim 51, wherein the cardiomyocytes, fibroblasts and / or endothelial cells are derived from the same ES cell. 52. The method of claim 51, wherein the cardiomyocytes, fibroblasts and / or endothelial cells are derived from different ES cells. The cardiac specific regulatory sequence is selected from the promoters of αMHC, MLC22v, MLC1a, MLC2a and βMHC, the fibroblast specific regulatory sequence is selected from the promoters of Tie2, Tie1 and caterin (original word: Catherin), and the endothelium 54. A method according to any of claims 51 to 53, wherein the cell specific regulatory sequence is selected from the promoters of collagen I promoter. 55. Any of the cardiomyocytes, fibroblasts and / or endothelial cells, wherein the reporter is individually selected from enhanced green fluorescent protein ECFP (blue), EYFP (yellow) and hcRFP (red) The method described in 1. 56. The method according to any of claims 51 to 55, wherein the resistance gene and the reporter are interrupted by an internal ribosome entry site (IRES). 57. A vector or vector composition comprising the recombinant nucleic acid molecule of any of claims 51-56. 58. A cell or a plurality of cells comprising the vector or vector composition of claim 57. 45. An array comprising a solid support and the cells of claim 43, the cell aggregate of claim 44, or the tissue of claim 45 attached thereto or suspended therefrom. 60. The array of claim 59, which is a microelectrode array (MEA). 61. An apparatus for analyzing an array according to claim 59 or 60. (A) the cell according to claim 43, the cell aggregate according to claim 44, the tissue according to claim 45, the organ according to claim 46, or the array according to claim 59 or 60; Contacting a test sample comprising a test substance;
(B) determining a phenotypic response in the test sample when compared to the control sample, the change in phenotypic response in the test sample when compared to the control sample is Obtaining a test substance capable of influencing cell generation and / or tissue structure formation, comprising the step of being an indicator that the test substance has an effect on cell generation and / or tissue structure formation, and / or A way to profile. The test sample is contacted with the test substance before, during, or after the cell or cell aggregate is subjected to the method according to any one of claims 1 to 14 or 21 to 42. 64. The method of claim 62, wherein: 64. The method of claim 62 or 63, wherein the contacting step further comprises contacting the test sample with at least one second test substance in the presence of the first test substance. The method according to any one of claims 62 to 64, wherein the test substance is contained and provided as a collection of test substances, preferably in a first screening. 66. The method of claim 65, wherein the collection of test substances has a diversity of about 10 ^{3 to} about 10 ⁵ species. 68. The method of claim 66, wherein the diversity of the collection of test substances is decreased sequentially. 68. A method according to any of claims 61 to 67, performed on an array according to claim 59 or 60. 69. A method according to any of claims 61 to 68, wherein the phenotypic response comprises electrophysiological properties during an ongoing differentiation process. 70. The method of any one of claims 1-14, 21-42 or 62-69, wherein the one or more cells are genetically engineered to (over) express or inhibit expression of a target gene. 71. A method according to any of claims 1 to 14, 21 to 42 or 62 to 70, wherein a compound known to activate or inhibit differentiation processes and / or tissue structure formation is added to the medium. 72. A method according to any of claims 1 to 14, 21 to 42 or 62 to 71, wherein the one or more cells or tissues are contained in a container. 73. A method according to any of claims 1 to 14, 21 to 42 or 62 to 72, comprising taking three or more measurements, optionally at different locations within the container. 74. A method according to any of claims 72 or 73, wherein the container is a well in a microtiter plate. 75. The method of claim 74, wherein the microtiter plate is a 24-, 96-, 384-, or 1586-well plate. 76. A method of manufacturing a drug comprising the steps of any of claims 62-75. 77. A method of manufacturing an agent that aids wound healing and / or healing of damaged tissue comprising the steps of any of claims 62-76. 77. further comprising altering, removing and / or derivatizing a portion of said substance that is predicted to increase toxicity, bioavailability, solubility and / or half-life. 77. The method according to 77. 79. A method according to any of claims 76 to 78, further comprising the step of mixing the isolated or modified material with a pharmaceutically acceptable carrier. 58. A vector or composition of vectors, pluripotent or useful according to any of claims 1 to 14, 21 to 42, 50 to 56 or 62 to 79, and A kit or composition comprising pluripotent cells and optionally a medium, a recombinant nucleic acid molecule, or a standard compound. 58. A cell according to claim 43, a cell aggregate according to claim 44, a tissue according to claim 45 or an organ according to claim 46, an implant or graft according to claim 47, a claim 57. 62. A vector or composition of vectors of claim 48, a composition of claim 48, an array of claim 59 or 60, or a device of claim 61 in drug discovery or pharmacokinetic or pharmacological profiling. .

Images